Virus-like particle conjugates for diagnosis and treatment of tumors

ABSTRACT

The present disclosure is directed to methods and compositions for the diagnosis and/or treatment of tumors, such as ocular tumors, using virus-like particles conjugated to photosensitive molecules.

RELATED APPLICATION

This application is a continuation of U.S. application Ser. No.17/395,369, filed Aug. 5, 2021, which is a continuation of U.S.application Ser. No. 16/778,361, now U.S. Pat. No. 11,110,181, filedJan. 31, 2020, which is a continuation of U.S. application Ser. No.16/143,147, now U.S. Pat. No. 10,588,984, filed Sep. 26, 2018, which isa continuation of U.S. application Ser. No. 15/023,169, now U.S. Pat.No. 10,117,947, filed Mar. 18, 2016, which is a national stage filingunder 35 U.S.C. § 371 of international application numberPCT/US2014/056412, filed Sep. 18, 2014, which was published under PCTArticle 21(2) in English and claims the benefit under 35 U.S.C. § 119(e)of U.S. provisional application Ser. No. 61/879,627, filed Sep. 18,2013, each of which is herein incorporated by reference in its entirety.

FIELD OF THE INVENTION

This disclosure relates to the field of tumor diagnostics andtherapeutics.

BACKGROUND OF THE INVENTION

Although numerous treatments are available for cancer, many forms ofcancer remain incurable, untreatable or become resistant to standardtherapies and effective treatments for many cancers have undesirableside effects. Ocular cancers, such as ocular melanoma andretinoblastoma, are particularly challenging to treat. A patientdiagnosed with ocular melanoma, depending on the size of the tumor, hasfew treatment options, including: (1) surgical procedures such asresection, enucleation or exenteration, all of which are highly invasiveand mainly involve the removal of the eye and part of the optic nerve(after surgery the patient is usually fitted for an artificial eye); and(2) plaque brachytherapy, a type of radiation therapy, where a thinpiece of metal (e.g., gold) with radioactive seeds covering one side issewn onto the outside wall of the eye with the seeds aimed at the tumor.The thin piece of metal is removed at the end of treatment, whichusually lasts for several days. Severe radioactive related complicationsinclude: cataract formation, which is the most common, followed byvitreous hemorrhage. Other complications include dry eye, keratitis,radiation-induced iris neovascularization, neovascular glaucoma,radiation-induced retinopathy, radiation-induced optic neuropathy,episcleral deposits, scleral necrosis and/or extraocular musclealterations. Radiation retinopathy has been reported to occur in 10-63%of patients treated with plaque brachytherapy, and the mean time fromtreatment to the development of maculopathy is approximately 25.6months.

SUMMARY OF THE INVENTION

The present disclosure provides, at least in in part, methods andcompositions for detecting and/or selectively targeting tumor cells, forexample, for the diagnosis and/or treatment of cancer (e.g., ocularcancer). In some instances, the methods and compositions provided hereincan be used to selectively kill cancerous tumor cells without damaginghealthy cells. For example, viral-like nanoparticles that comprise(e.g., are conjugated to) photosensitive molecules may be selectivelydelivered to tumor cells and photoactivated by exposure to light. Whenphotoactivated, a photosensitive molecule absorbs photons, and thatabsorbed energy produces molecular changes that cause toxicity (e.g.,cellular toxicity). A “photosensitive viral-like nanoparticle,” (alsoreferred to herein as a “photosensitive virus-like particle”) refers toa viral-like nanoparticle conjugated to a photosensitive molecule.Surprisingly, conjugation of photosensitive molecules to viral-likenanoparticles does not interfere with the tissue/tumor tropism of thenanoparticles (e.g., the specificity of the viral-like nanoparticles fora particular host tumor tissue or tumor cell).

Viral-like nanoparticles (also referred to as virus-like particles(VLPs)) of the present disclosure, generally, are assembled from L1capsid proteins, or a combination of L1 and L2 capsid proteins, and thephotosensitive molecules, in some embodiments, are conjugated to acapsid protein that forms the viral-like nanoparticle. Thus, variousaspects of the disclosure provide tumor-targeting viral-likenanoparticles that comprise photosensitive molecules conjugated tocapsid proteins.

Some aspects of the disclosure also provide tumor-targeting virus-likeparticles that comprise about 50 to about 500, about 50 to about 600,about 50 to about 700, about 50 to about 800, about 50 to about 900, orabout 50 to about 1000 photosensitive molecules per particle. In someembodiments, tumor-targeting virus-like particles comprise about 400,about 500, about 600, about 700, about 800, about 900 or about 1000photosensitive molecules per particle. In some embodiments,tumor-targeting virus-like particles comprise 500 photosensitivemolecules or 1000 photosensitive molecules per particle.

In some embodiments, the capsid proteins are papilloma virus capsidproteins. For example, in some embodiments, the papilloma virus capsidproteins are non-human papilloma virus capsid proteins, such as bovinepapilloma virus capsid proteins. In some embodiments, the virus-likeparticles comprise human papilloma virus capsid proteins and do notcross-react with human papilloma virus (HPV) 16, HPV 18 or pre-existingantibodies specific for HPV.

In some embodiments, the virus-like particles comprise papilloma L1 orL1/L2 proteins (e.g., of human, bovine, or other species). In someembodiments, the L1 or L1/L2 VLPs do not cross-react with neutralizingantibodies to human papilloma virus (HPV) 16, HPV 18 or pre-existingantibodies specific for other HPVs. However, in some embodiments, thevirus-like particles comprise human papilloma virus capsid proteins ofHPV16.

In some embodiments, the photosensitive molecules are conjugated tosurface-exposed peptides of capsid proteins.

In some embodiments, the virus-like particles comprise L1 capsidproteins or a combination of L1 and L2 capsid proteins. In someembodiments, the virus-like particles consist of L1 capsid proteins.

In some embodiments, a virus-like particle comprises BPV L1 capsidprotein (e.g., SEQ ID NO: 2), a combination of BPV L1 and BPV L2 capsidproteins. In some embodiments, a virus-like particle comprises HPV L1capsid proteins, or a combination of HPV L1 and HPV L2 capsid proteins.In some embodiments, the HPV L1 capsid protein is a variant HPV16/31 L1protein having modified immunogenicity and/or antigenicity (e.g., SEQ IDNO: 1). Thus, in some embodiments, a virus-like particle comprises orconsists of variant HPV16/31 L1 capsid proteins or a combination ofvariant HPV16/31 L1 capsid proteins (e.g., SEQ ID NO: 1) and HPV L2capsid proteins.

In some embodiments, the capsid proteins of a virus-like particle havemodified immunogenicity and/or antigenicity. A non-limiting example ofsuch a capsid protein is HPV16/31 L1 capsid proteins (e.g., SEQ ID NO:1). Virus-like particles that contain modified capsid proteins may bereferred to herein as virus-like particles that contain modifiedimmunogenicity and/or antigenicity compared to wild-type virus-likeparticles.

In some embodiments, the photosensitive molecules are covalentlyconjugated to capsid proteins. In some embodiments, the photosensitivemolecules are conjugated to an amino acid of the capsid proteins. Insome embodiments, the photosensitive molecules are conjugated to anamine group (e.g., primary aliphatic amine) of an amino acid of thecapsid proteins. In some embodiments, the photosensitive molecules areconjugated to amine groups of lysine residues (e.g., side chain amine oflysine) of the capsid proteins. In some embodiments, the photosensitivemolecules are conjugated to amine groups of arginine and/or histidineresidues) of the capsid proteins. The present disclosure providesmethods for conjugating photosensitive molecules to lysine and otheramino acids that contain amine groups.

In some embodiments, the photosensitive molecules do not compromise(e.g., prevent, interfere with or inhibit) binding of the virus-likeparticle to the surface of tumor cells. In some embodiments, thephotosensitive molecules do not compromise (e.g., prevent, interferewith or inhibit) binding of the virus-like particle to heparan sulphateproteoglycans or other polysaccharides on the surface of tumor cells.

In some embodiments, the virus-like particles comprise about 10 to about1000 photosensitive molecules. In some embodiments, the virus-likeparticles comprise about 50 to about 1000 photosensitive molecules. Insome embodiments, the virus-like particles comprise about 100 to about1000 photosensitive molecules. In some embodiments, the virus-likeparticles comprise about 100 to about 500 photosensitive molecules. Insome embodiments, the virus-like particles comprise about 500 to about1000 photosensitive molecules, or more.

In some embodiments, the virus-like particles comprise about 10 to about1000 photosensitive molecules that are conjugated to lysine residues orother amino acid residues of L1 capsid proteins, L2 capsid proteins, ora combination of L1 capsid proteins and L2 capsid proteins.

In some embodiments, the photosensitive molecules are activated byinfrared, near-infrared or ultraviolet light. A photosensitive moleculeis considered to be “activated” when the molecule absorbs photons, andthat absorbed energy produces molecular changes that cause toxicity, asdescribed elsewhere herein.

In some embodiments, the photosensitive molecules comprise a fluorescentdye, an infrared dye, a near infrared dye, a porphyrin molecule, achlorophyll molecule, or a combination of any two or more of theforegoing.

In some embodiments, the photosensitive molecules are porphyrinmolecules. Examples of porphyrin molecules for use in accordance withthe present disclosure include, without limitation, HpD (hematoporphyrinderivative), HpD-based, BPD (benzoporphyrin derivative), ALA(5-aminolevulinic acid) and texaphyrins. In some embodiments, theporphyrin molecule is verteporfin (Visudyne®)

In some embodiments, the photosensitive molecules are chlorophyllmolecules. Examples of chlorophyll molecules for use in accordance withthe present disclosure include, without limitation, chlorins, purpurinsand bacteriochlorins.

In some embodiments, the photosensitive molecules are dyes. Examples ofdyes for use in accordance with the present disclosure include, withoutlimitation, phthalocyanine and naphthalocyanine.

In some embodiments, the phthalocyanine dye is both a fluorescentmolecule and a near infrared molecules. For example, in someembodiments, the phthalocyanine dye is IR700 dye (e.g., IRDye® 700DX,LI-COR®). An IR700 dye is a fluorescent dye that has an absorption andemission wavelengths in the near-infrared (NIR) spectrum typicallybetween 680 nm and 800 nm. Other fluorescent dyes having an absorptionand emission wavelengths in the NIR spectrum are provided herein.

In some embodiments, photosensitive molecules are selected fromphthalocyanine dyes (e.g., IR700 dye such as IRDye® 700DX), porphyrinmolecules (e.g., verteporfin such as Visudyne®) and a combination ofphthalocyanine dyes and porphyrin molecules.

Some aspects of the disclosure provide methods that compriseadministering, to a subject having a tumor, any one of the virus-likeparticles, or photosensitive virus-like particles, provided herein. Insome embodiments, the methods comprise activating the photosensitivemolecules of a virus-like particle at a wavelength of light that permitsvisualization of the light sensitive molecules. Thus, in someembodiments, the photosensitive molecules of the present disclosure areused as imaging agents and/or diagnostic agents. In some embodiments,the methods comprise activating the photosensitive molecules at awavelength of light that causes the molecule to be cytotoxic. In someembodiments the methods comprise activating the photosensitive moleculesat a wavelength of light generating an energy transfer within the tumorcell that creates direct and irreversible cell damage leading tonecrosis. Thus, in some embodiments, the photosensitive molecules of thepresent disclosure are used as therapeutic and/or prophylactic agents.

Some aspects of the disclosure provide methods that compriseadministering, to a subject having a tumor, a tumor-targeting virus-likeparticle comprising photosensitive molecules conjugated to capsidproteins. In some embodiments, the methods comprise activatingphotosensitive molecules of the virus-like particles at a wavelengththat renders the molecules visible. That is, the photosensitivemolecules re-emit light upon light excitation. In some embodiments, themethods comprise activating photosensitive molecules at a wavelengththat renders the molecules cytotoxic, thereby killing cells of thetumor. That is, the photosensitive molecules undergo a molecular changeupon light excitation that results in the photosensitive moleculesbecome toxic to cells.

Some aspects of the disclosure provide methods that compriseadministering, to a subject having a tumor, a tumor-targeting virus-likeparticle comprising about 50 to about 1000, about 50 to 500, or about500 to 1000 photosensitive molecules. In some embodiments, methodscomprise administering, to a subject having a tumor, a tumor-targetingvirus-like particle comprising about 100, 200, 300, 400, 500, 600, 700,800, 900, 1000 or more photosensitive molecules. In some embodiments,the methods comprise activating photosensitive molecules at a wavelengththat renders the molecules visible. In some embodiments, the methodscomprise activating photosensitive molecules at a wavelength thatrenders the molecules cytotoxic, thereby killing cells of the tumor.

In some embodiments, the photosensitive molecules are laser activated.In some embodiments, the laser is an infrared, near-infrared orultraviolet laser. In some embodiments, the infrared laser is 5 Joules(J) to 100 J (or J/cm²) (e.g., 5 J, 6 J, 7 J, 8 J, 9 J, 10 J, 11 J, 12J, 13 J, 14 J, 15 J, 16 J, 17 J, 18 J, 19 J, 20 J, 21 J, 22 J, 23 J, 24J, 25 J, 26 J, 27 J, 28 J, 29 J, 30 J, 31 J, 32 J, 33 J, 34 J, 35 J, 36J, 37 J, 38 J, 39 J, 40 J, 41 J, 42 J, 43 J, 44 J, 45 J, 46 J, 47 J, 48J, 49 J, 50 J, 51 J, 52 J, 53 J, 54 J, 55 J, 56 J, 57 J, 58 J, 59 J, 60J, 61 J, 62 J, 63 J, 64 J, 65 J, 66 J, 67 J, 68 J, 69 J, 70 J, 71 J, 72J, 73 J, 74 J, 75 J, 76 J, 77 J, 78 J, 79 J, 80 J, 81 J, 82 J, 83 J, 84J, 85 J, 86 J, 87 J, 88 J, 89 J, 90 J, 91 J, 92 J, 93 J, 94 J, 95 J, 96J, 97 J, 98 J, 99 J or 100 J (or J/cm²)). In some embodiments, the laseris applied for about 5 seconds to about 5 minutes.

In some embodiments, the photosensitive molecules are activated at about30 minutes to about 48 hours after administering the virus-likeparticles to a subject. For example, the photosensitive molecules may beactivated at 30 minutes after administering the virus-like particles toa subject. In some embodiments, the photosensitive molecules areactivated 1 hour, 2 hours (h), 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h,11 h, 12 h, 13 h, 14 h, 15 h, 16 h, 17 h, 18 h, 19 h, 20 h, 21 h, 22 h,23 or 24 h after administering the virus-like particle to a subject. Insome embodiments, the photosensitive molecules are activated 1 day, 2days or 3 days after administering the virus-like particle to a subject.

In some embodiments, the tumor is an ocular tumor or a tumor that hasmetastasized to the eye. For example, in some embodiments, the oculartumor is located in the vitreous, choroidal space, iris, ciliary body,sclera, fovea, retina, optic disk or optic nerve.

In some embodiments, the tumor is located in a lung, pleura, liver,pancreas, stomach, esophagus, colon, breast, ovary, prostate, brain,meninges, testis, gastrointestinal tract, kidneys or bladder.

In some embodiments, the tumor is accessible without surgicalintervention.

In some embodiments, the tumor is located in the head, neck, cervix,larynx or skin.

In some embodiments, the tumor is an orphan or rare disease.

In some embodiments, the tumor is cancerous. In some embodiments, thetumor is metastatic. In some embodiments, the tumor is pre-cancerous ordysplastic.

In some embodiments, the virus-like particles are administered byinjection. For example, the virus-like particles may be administered byinjection intraocularly, into the vitreous, or intravenously. In someembodiments, the virus-like particles are administered with a hollow orcoated needle, mini-needle or micro-needle. In some embodiments, thevirus-like particles are administered topically. In some embodiments,the virus-like particles are administered by implantation.

In some embodiments, the capsid proteins are papilloma virus capsidproteins. For example, in some embodiments, the papilloma virus capsidproteins are non-human papilloma virus capsid proteins, such as bovinepapilloma virus (BPV) capsid proteins. In some embodiments, thevirus-like particles comprise human papilloma virus capsid proteins anddo not cross-react with human papilloma virus (HPV) 16, HPV 18 orpre-existing antibodies specific for HPV. In some embodiments, thevirus-like particles comprise human papilloma virus type 16 capsidproteins. In some embodiments the VLPs do not bind antibodies specificfor human papilloma virus (HPV) 16, HPV 18 VLPs or pre-existingantibodies specifically induced by HPV infection.

Some aspects of the disclosure provides methods of detecting, in asubject, tumors (e.g., ocular tumors and malignant nevi), the methodscomprising administering to the subject (e.g., to the eye of thesubject) any one of the virus-like particle provided herein, such as avirus-like particle comprising a photosensitive molecule (e.g.,fluorescent dye or infrared dye), and detecting the location of thetumor. In some embodiments, the methods comprise detecting the locationof the tumor by illuminating the subject (e.g., eye of the subject) witha laser (e.g., ultra-violet or infrared laser). In some embodiments, themethods comprise identifying the subject suspected of having a tumorbefore administering the virus-like particle. In some embodiments, themethods comprise diagnosing and/or treating the tumor by administeringphotosensitive virus-like particles to a tumor of the subject or to thea subject having or suspected of having a tumor.

Other aspects of the disclosure provide methods of selectivelyinhibiting proliferation or killing of cancerous cells withoutinhibiting proliferation or viability of non-cancerous (e.g., normal,healthy) cells, the methods comprising administering to a tumor of asubject (e.g., to an ocular tumor of the subject) any one of thetumor-targeting virus-like particles provided herein, such as virus-likeparticles comprising photosensitive molecules (e.g., infrared dye), andirradiating cancerous cells of the tumor by subjecting the tumor to aninfrared laser (e.g., at a wavelength of about 660 nm to 740 nm and at adose of at least 8 Joules), effectively.

In some embodiments, the present disclosure provides a viral-likenanoparticle (also referred to as a virus-like particle) comprisingphotosensitive molecules conjugated to papilloma virus L1 proteins(e.g., bovine papilloma virus L1 proteins). In some embodiments, theviral-like nanoparticles are 20 to 60 nanometers (e.g., 10, 25, 30, 35,40, 45, 50, 55 or 60 nanometers) in diameter. In some embodiments, aviral-like nanoparticle contains 300 to 500 L1 (e.g., BPV L1) capsidproteins, for example 360 L1 capsid proteins (e.g., based on icosahedralsymmetry). It should be appreciated that in some embodiments, viral-likenanoparticles each contain about 300, 310, 320, 330, 340, 350, 360, 370,380, 390, 400, 410, 420, 430, 440, 450, 460, 470, 480, 490 or 500 L1(e.g., BPV L1) capsid proteins. However, in some embodiments, aviral-like nanoparticle contains less than 300 L1 (e.g., BPV L1) capsidproteins.

In some embodiments, the present disclosure provides a bovine papillomavirus viral-like nanoparticle covalently conjugated to 100 to 1000photosensitive molecules (e.g., 100, 200, 300, 400, 500, 600, 700, 800,900 or 1000 molecules). In some embodiments, the capsid proteins of thebovine papilloma virus (BPV) viral-like nanoparticle comprise or consistof BPV L1 capsid proteins or a combination of BPV L1 and BPV L2 capsidproteins. In some embodiments, the photosensitive molecules areconjugated to the viral-like nanoparticles (or to capsid proteins of theviral-like nanoparticles) through a covalent bond formed by reacting anester group in the photosensitive molecules with an amine group in thecapsid proteins, thereby forming an amide bond. Thus, in someembodiments, capsid proteins of viral-like nanoparticles of the presentdisclosure are conjugated to photosensitive molecules through amidebonds.

In some embodiments, the present disclosure provides a viral-likenanoparticle comprising 300 to 500 BPV L1 capsid proteins and/or adiameter of 20 60 nm, at least some of which (e.g., 10%, 20%, 30%, 40%,50%, 60%, 70%, 80%, 90% or 100%) are covalently conjugated (e.g.,through an amide bond) to 1 to 5 (e.g., 1, 2, 3, 4 or 5) photosensitivemolecules (e.g., IR700 dye such as IRDye® 700DX). The present disclosurealso provide methods of producing viral-like nanoparticles and methodsof administering viral-like nanoparticles to a subject as a diagnostic,therapeutic or prophylactic agent.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows a mechanism for inducing cell death using a virus-likeparticle (VLP) conjugated to a photosensitive molecule.

FIG. 2 shows a comparison of bivalent targeting. e.g., by an antibody,and multivalent targeting. e.g., by a VLP.

FIG. 3 shows a graph demonstrating that specificity of VLP binding tocells is mediated by heparan sulfate proteoglycan (HSPG) interactionsand is inhibited by heparin. It further shows specific killing of tumorcells only when the photosensitive VLPs are bound to the cell and thecells subjected in infrared irradiation.

FIG. 4 shows a graph demonstrating that cell death depends on the doseof infrared radiation and the amount of the VLP and photosensitivemolecule (e.g., dye) delivered.

FIG. 5 shows a graph demonstrating in vitro ovarian cancer cell (SKOV-3)death upon irradiation with VLPs (designated PsV in the figure)conjugated to IR700.

FIG. 6A shows an electrospray ionization-time-of-flight (ESI-TOF)analysis of control VLPs. FIG. 6B shows an ESI-TOF analysis of VLPs(designated PsV in the figure) conjugated to 1000 molecules of IR700.

FIGS. 7A-7C show graphs of cell death of a human epidermal growth factorreceptor 2 negative (HER2⁻) ocular melanoma cell line (92.1), comparingthe effectiveness of bivalent agents (e.g., antibodies) and multivalentagents (e.g., photosensitive VLPs, also referred to as VLP conjugates,designated PsV in the figure).

FIGS. 8A-8C show graphs of cell death of an human epidermal growthfactor receptor 2 positive (HER2⁺) ovarian cancer cell line (SKOV-3),comparing the effectiveness of bivalent agents (e.g., antibodies) andmultivalent agents (e.g., photosensitive V LPs, also referred to as VLPconjugates, designated PsV in the figure).

FIG. 9 shows a graph demonstrating vaccine induced anti-HPV16neutralizing antibodies do not block binding of BPV*IR700 VLPs to theocular melanoma cell line, 92.1.

FIG. 10A shows a chemical structure of IRDye® 700DX NHS ester. FIG. 10Bshows a chemical structure of Visudyne® with a reactive carboxyl groupcircled.

FIG. 11 shows a reaction scheme involving(1-ethyl-3-(-3-dimethylaminopropyl) carbodiimide hydrochloride) (EDC)and sulfo-N-Hydroxysuccinimide (sulfo-NHS) mediated linking of Visudyne®and VLP. In this scheme, {circle around (1)} represents Visudyne® and{circle around (2)} represents VLP. Note that there are 2 routes to thedesired end product. The presence of sulfo-NHS tends to stabilize thereaction and enhances the production of the desired product.

FIG. 12 shows histograms of representative samples of the HSPG-dependentbinding of viral-like nanoparticles containing HPV16 capsid proteins,variant HPV16/31 capsid proteins and BPV1 capsid proteins (L1, or L1 andL2 proteins) binding to various types of cancer cells.

FIGS. 13A and 13B shows images of excised tumor tissue in bright field(FIG. 13A) and fluorescence (FIG. 13B) from PBS-injected negativecontrol mice at 12 hours, photosensitive viral-likenanoparticle-injected mice at 12 hours (#3 and #4) and photosensitiveviral-like nanoparticle-injected mice at 24 hours (#1 and #2) followinginjection.

FIG. 14 shows a quantitative representation of total tumor associatedviral-like nanoparticle-related fluorescence in ex vivo TC-1 tumorsamples excised 12 and 24 hrs after intravenous injection of the VLPs(same tumors as FIG. 13).

FIG. 15 shows a schematic of the experimental design for Example 14.

FIGS. 16A and 16B show graphs of percentage of cell death after in vivoadministration of photosensitive viral-like nanoparticles (designatedNPs in the figure) and light titration on subcutaneous 92.1 ocularmelanoma (OM) cells (cell viability measured 24 hours after lighttreatment).

FIGS. 17A-17C show raw histograms for data presented in FIGS. 16A and16B.

FIG. 18 (top panel) shows tissue samples obtained from animalsinoculated subcutaneously with 2×10 TC-1 tumor cells in 100 μl of PBSand administered: (1) no treatment, (2) 100 μg viral-like nanoparticles(designated NPs in the figure) assembled from variant HPV16/31 L1proteins and HPV L2 proteins, labeled with IRDye® 700DX [without light,(3) PBS with 50 J/cm² light, (4) 200 μg viral-like nanoparticles with 50J/cm² light, (5) 100 μg viral-like nanoparticles with 50 J/cm² light and(6) 50 μg viral-like nanoparticles with 50 J/cm² light. FIG. 18 (bottompanel) shows percentage of dead cells for each of the six testconditions.

FIG. 19A shows a schematic of the experiment described in Example 15.FIG. 19B shows a graph of percent survival in animals injected withviral-like nanoparticles (designated nanoparticles in the figure) versuscontrol (with light). FIG. 19C shows tumor volume (top panel), “E7tetramer⁺ CD8⁺ T-cells” and “INF-gamma secreting CD8⁺ cells” inindividual mice.

FIG. 20 shows a graph of results from a potency assay, comparing theeffects of photosensitive BPV viral-like nanoparticles andphotosensitive HPV viral-like nanoparticles on cell viability.

FIG. 21 shows a graph of results form a binding assay, comparing bindingof photosensitive BPV viral-like nanoparticles and photosensitive HPVviral-like nanoparticles to cells.

FIG. 22 shows a graph of tumor growth curve of head and neck cancercells following treatment with photosensitive viral-like nanoparticles(designated PsV in the figure).

FIGS. 23A and 23B depict examples of a photosensitive viral-likenanoparticle production process of the present disclosure (e.g., asdescribed in Example 20).

DETAILED DESCRIPTION OF THE INVENTION

Photodynamic therapy (PDT) is a form of phototherapy using nontoxicphotosensitive molecules that, when selectively exposed to light, becometoxic, and target and/or kill, malignant and other diseased cells. Achallenge posed by PDT in the treatment of cancer is the delivery ofhigh concentrations of photosensitive molecules exclusively to tumorcells. To achieve targeted delivery, antibodies can be used, though theyare limited by their delivery capacity, which is in the range of 2-8photosensitive molecules per antibody. Further, there are importanttumors that lack an identified tumor receptor molecule and, thus, cannotbe targeted with an antibody. As a consequence, multiple tumors remainuntreatable (e.g., ocular melanoma). In addition, many of the molecules(e.g., EGFR) targeted by antibody/dye conjugates are also found on thesurface of non-tumor cells, leading to unwanted off target effects.

The present disclosure is based, in part, on the unexpected discoverythat virus-like particles (VLPs) (e.g., papilloma VLPs) (also referredto herein as viral-like nanoparticles) can be chemically modified tocarry many photosensitive molecules (e.g., IR700) without losing theirtumor-targeting capability or structural stability. For example, in someembodiments, VLPs can be chemically modified to carry more than 50molecules, more than 100 molecules, or more than 1000 molecules (orabout 1000 photosensitive molecules). Virus-like particles assembledfrom L1, or L1 and L2 capsid proteins, can selectively bind to andinfect cancer cells without affecting non-cancerous cells, therebyminimizing the cytotoxicity of treatments (see U.S. Patent ApplicationPublication No. US20100135902A1, the entirety of which is incorporatedby reference herein). Further, in some instances, the delivery of highamounts of photosensitive molecules per particle enables the selectivekilling of tumor cells upon light radiation with extremely small amountsof drug (e.g., picomolar concentrations).

A key cell binding characteristic of a VLP is the presence of a highnumber of heparin binding sites on the capsid proteins (e.g., L1).Conjugation of photosensitive molecules to surface amino acids (e.g.,conjugation via an amide bond to surface amino acids such as surfacelysine residues, arginine residues and histidine residues),surprisingly, does not compromise binding of the VLP to heparan sulphateproteoglycans (HSPGs) on the surface of tumor cells. Although, thepresent disclosure describes conjugation of photosensitive molecules tosurface-exposed peptides of capsid proteins, it should be understoodthat photosensitive molecules may be conjugated to any peptides ofcapsid proteins. That is, photosensitive molecules may be conjugated toL1 proteins only or to a combination of L1 and L2 proteins. The proteinand amino acid residue to which a photosensitive molecule is conjugatedcan depend on the composition of the virus-like particle.

The foregoing discoveries have important implications for thedevelopment of novel targeted cancer treatments. For example, thephotosensitive VLPs (also referred to as VLP conjugates) of the presentdisclosure provide an advantage relative to other targeting moleculessuch as antibodies, which have a very limited delivery capacity. Inaddition, the photosensitive VLPs of the present disclosure are usefulfor targeting a wide range of tumors that otherwise cannot be targetedby antibodies or other targeting molecules (e.g., ocular tumors) becausesuitable tumor-surface specific determinants have not been identified.Further, the photosensitive VLPs are useful for treating distantmetastases. In addition the photosensitive VLPs are useful for diagnosisand treatment of early malignant or pre-cancerous lesions (e.g., ocularnevi that are transformed, pre-malignant or malignant).

A “virus-like particle” (VLP), as used herein, refers to an organizedcapsid-like structure (e.g., roughly spherical or cylindrical in shape)that comprises self-assembling ordered arrays of L1 or L1 and L2capsomers and does not include a viral genome. Virus-like particles aremorphologically and antigenically similar to authentic virions, but theylack viral genetic material (e.g., viral nucleic acid), rendering theparticles non-infectious. A VLP may be used to deliver to a recipientcell an agent (e.g., prophylactic agent, therapeutic agent or diagnosticagent) or an enclosed circular or linear DNA or RNA molecule. It shouldbe understood that the terms “virus-like particle,” or “VLP” and“pseudovirus,” or “PsV” may be used interchangeably herein and may alsobe used interchangeably with the term “viral-like nanoparticle.”

A “tumor-targeting virus-like particle,” as used herein, refers to a VLPthat targets tumor (e.g., cancerous) cells without targeting non-tumor(e.g., non-cancerous, otherwise normal, healthy) cells (e.g., in intacttissue).

VLPs in accordance with the present disclosure may have a modifiedimmunogenicity and/or antigenicity with respect to the wild typepapillomavirus VLPs. The VLPs may, for example, be assembled fromcapsomers having a variant capsid protein with modified immunogenicityand/or antigenicity. A variant capsid protein with “modifiedimmunogenicity and/or antigenicity” is one that is modified naturally orsynthetically (e.g., mutated, substituted, deleted, pegylated orinserted) at an amino acid to reduce or prevent recognition of thecapsid protein by pre-existing (e.g., endogenous) viralserotype-specific antibodies. A variant capsid protein may be a humanpapillomavirus (HPV) L1 variant, a non-human papillomavirus L1 variant,or a papillomavirus L1 variant based on a combination of amino acidsfrom different HPV serotypes. For example, an L1 variant with modifiedimmunogenicity and/or antigenicity may be a recombinant protein based onHPV serotype 16 and HPV serotype 31 (referred to herein as a “variantHPV16/31 L1 protein”—SEQ ID NO: 1), which is described in InternationalPub. No. WO/2010/120266, the entirety of which is incorporated byreference herein.

In some embodiments, a VLP is a papilloma virus VLP. The VLP may be ahuman papilloma virus VLP (e.g., derived from a virus that can infecthuman), while in other embodiments, the VLP is a non-human papillomavirus VLP. Examples of non-human VLPs include those derived from,without limitation, bovine papilloma viruses, murine papilloma viruses,cotton-rabbit papilloma viruses and macaque or rhesus papilloma virusparticles. In some embodiments, the VLPs are bovine papilloma virusviral-like nanoparticles (e.g., type 1 viral-like nanoparticles) (e.g.,assembled from BPV L1 capsid proteins or a combination of BPV L1 and BPVL2 capsid proteins).

A “capsid protein,” as used herein, refers to a protein monomer, severalof which form a capsomer oligomer. A “capsomer,” as used herein, refersto the basic oligomeric structural unit of a viral capsid, which is anouter covering of protein that protects the genetic material of a virussuch as, for example, human papillomavirus (HPV). The capsid proteins ofthe present disclosure include papillomavirus L1 major capsid proteinsand papillomavirus L2 minor capsid proteins. In some embodiments, theVLPs of the present disclosure contain only L1 capsid proteins, while inother embodiments, the VLPs contain a mixture (or combination) of L1 andL2 capsid proteins.

In some embodiments, the percentage of L1 capsid proteins in avirus-like particle is greater than the percentage of L2 capsid proteinsin the virus-like particle. For example, in some embodiments, thepercentage of L1 capsid proteins in a virus-like particle is 80% to 100%(of the total number of capsid proteins in the virus-like particle). Insome embodiments, the percentage of L1 capsid proteins in a virus-likeparticle is 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%or 100%. In some embodiments, the percentage of L2 capsid proteins in avirus-like particle is 1% to 25% (of the total number of capsid proteinsin the virus-like particle). For example, some embodiments, thepercentage of L2 capsid proteins in a virus-like particle is 1%, 2%, 3%,4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%or 20%.

In some embodiment, a virus-like particle contains 12 to 72 L2 proteins.In some embodiment, a virus-like particle contains 360 L1 proteins and12 to 72 L2 proteins. In some embodiments, capsid proteins assemble intoviral-like nanoparticles having a diameter of 20 to 60 nm. For example,capsid proteins may assemble into viral-like nanoparticles having adiameter of 20, 25, 30, 35, 40, 45, 50, 55 or 60 nm.

An “external capsid protein,” as used herein, refers to a capsid proteinthat is exposed at the surface of a VLP. In some embodiments, externalcapsid proteins (e.g., L1 proteins) are conjugated to a (e.g., at leastone) photosensitive molecule.

A “photosensitive molecule,” as used herein, refers to a nontoxicmolecule that, when exposed selectively to light, becomes “activated”(also referred to as “photoactivated”). In some embodiments, anactivated photosensitive molecule re-emits light upon light excitation(e.g., a fluorophore). In some embodiments, an activated photosensitivemolecule can become toxic, or can produce toxic molecules, upon lightexcitation. For example, a class of photosensitive molecules, referredto as photosensitizers, can be promoted to an excited state uponabsorption of light and undergo intersystem crossing with oxygen toproduce singlet oxygen. This singlet oxygen rapidly attacks any organiccompounds it encounters, thus is highly cytotoxic.

In accordance with various aspects of the present disclosure,photosensitive molecules may be conjugated to capsid proteins (e.g., L1and/or L2 capsid proteins) of the VLPs. In some embodiments, thephotosensitive molecules are covalently conjugated to capsid proteins ofthe VLPs. In some embodiments, the photosensitive molecules arecovalently conjugated to lysine residues of capsid proteins of the VLPs.VLPs that are conjugated to photosensitive molecules may be referred toherein as “VLP conjugates” or “photosensitive VLPs.” In someembodiments, the photosensitive molecules comprise an NHS(N-Hydroxysuccinimide) ester group that reacts with an amine group ofthe capsid protein (e.g., amine group of lysine or other amino acid) toform a covalent amide bond.

The ratio of photosensitive molecule (PM) to VLP may vary.

In some embodiments the ratio of VLP:PM is about 1:10 to about 1:1000,about 1:10 to about 1:500, about 1:50 to about 1:500, or about 1:50 toabout 1:1000. That it, in some embodiments, a VLP may comprise about 10to about 1000 photosensitive molecules. In some embodiments, the ratioof VLP:PM is 1:10, 1:15, 1:20, 1:25, 1:50, 1:75, 1:100, 1:150, 1:200,1:250, 1:300, 1:350, 1:400, 1:450, 1:500, 1:550, 1:600, 1:650, 1:700,1:750, 1:800. 1:850, 1:900, 1:950 or 1:1000. In some embodiments, theVLP may comprise 10, 15, 20, 50, 75, 100, 150, 200, 250, 300, 350, 400,450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 or 1000photosensitive molecules. In some embodiments, the VLP may comprise morethan 1000 photosensitive molecules or less than 10 photosensitivemolecules.

More than one photosensitive molecule may be conjugated to a singlecapsid protein. For example, a single capsid protein (e.g., L1 or L2capsid protein) may be conjugated to 1 to 5 (e.g., 1, 2, 3, 4 or 5)photosensitive molecules. Thus, more than one amino acids of a capsidprotein may be conjugated to a photosensitive molecule. In someembodiments, a single capsid protein may be conjugated to 1 to 2, 1 to3, or 2 to 3 photosensitive molecules. Thus, a photosensitive moleculemay be conjugated to 1, 2, 3, 4 or 5 different amino acids (e.g.,lysine, arginine and/or histidine, or other amino acid) of a singlecapsid protein.

Examples of photosensitive molecules for use in accordance with thepresent disclosure include, without limitation, fluorescent dyes,infrared dyes, near infrared dyes, porphyrin molecules and chlorophyllmolecules.

Examples of fluorescent dyes for use in accordance with the presentdisclosure include, without limitation, acridine orange, acridineyellow, Alexa Fluor, 7-Aminoactinomycin D,8-Anilinonaphthalene-1-sulfonic acid, ATTO dyes, auramine-rhodaminestain, benzanthrone, bimane, 9,10-Bis(phenylethynyl)anthracene,5,12-Bis(phenylethynyl)naphthacene, bisbenzimide, blacklight paint,calcein, carboxyfluorescein, carboxyfluorescein diacetate succinimidylester, carboxyfluorescein succinimidyl ester,1-chloro-9,10-bis(phenylethynyl)anthracene,2-chloro-9,10-bis(phenylethynyl)anthracene,2-chloro-9,10-diphenylanthracene, coumarin, DAPI, dark quencher, DiOC6,DyLight Fluor, Fluo-3, Fluo-4, FluoProbes, fluorescein, fluoresceinisothiocyanate, fluorescence image-guided surgery, fluoro-jade stain,fura-2, fura-2-acetoxymethyl ester, GelGreen, GelRed, green fluorescentprotein, heptamethine dyes, Indian yellow, Indo-1, Lucifer yellow,luciferin, MCherry, Merocyanine, Nile blue, Nile red, opticalbrightener, perylene, phloxine, phycobilin, phycoerythrin,phycoerythrobilin, propidium iodide, pyranine, rhodamine, rhodamine 123,Rhodamine 6G, RiboGreen, RoGFP, rubrene, (E)-stilbene, (Z)-stilbene,sulforhodamine 101, sulforhodamine B, SYBR Green I, synapto-pHluorin,tetraphenyl butadiene, tetrasodium tris(bathophenanthrolinedisulfonate)ruthenium(II), Texas Red, Titan yellow, TSQ, umbelliferone,yellow fluorescent protein and YOYO-1.

Examples of photosensitizing dyes for use in accordance with the presentdisclosure include, without limitation, HpD, Porfimer sodium(Photofrin®, Photogem®, Photosan Hemporfin®), m-THPC, Temoporfin(Foscan®), Verteporfin (Visudyne®), HPPH (Photochlor®),Palladium-bacteria-pheophorbide (Tookad®) 5-ALA, 5 aminolevulinic acid(Levulan®), 5-ALA methylester (Metvix®), 5-ALA benzylester (Benzvix®),5-ALA hexylester (Hexvix®), lutetium (III)-texaphyrin orMotexafin-lutetium (Lutex®, Lutrin®, Angrin®, Optrin®), SnET2, Tin (IV)ethyl etiopurpurin (Purlytin®, Photrex®), NPe6, mono-L-aspartyl chlorinee6, talaporfin sodium (Talporfin®, Laserphyrin®), BOPP, boronatedprotoporphyrin (BOPP®), Zinc phthalocyanine (CGP55847®), siliconphthalocyanine (Pc4®), mixture of sulfonated aluminium phthalocyaninederivatives (Photosens®), ATMPn, Acetoxy-tetrakis(beta-methoxyethyl-)porphycene), TH9402 and dibromorhodamine methylester.

Examples of photosensitizing dyes for use in accordance with the presentdisclosure include those that can be used in fluorescence imaging (e.g.,near infrared (NIR) fluorescent dyes) such as La Jolla Blue® and IRDye®700DX.

The present disclosure also provides methods of administering, to asubject having a tumor, a tumor-targeting virus-like particle comprisingphotosensitive molecules conjugated to capsid proteins, oradministering, to a subject having a tumor, a tumor-targeting virus-likeparticle comprising about 50 to about 1000 (e.g., 50, 100, 150, 200,250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900,950, or 1000) photosensitive molecules.

In some embodiments, the subject is a mammal, such as a human.

The mode of administration can be by injection, infusion, implantation,topical administration, or by any other means typically used to delivervirus-like particles. In some embodiments, hollow needles, coatedneedles, mini-needles or micro-needles are used, depending on the areaof injection. In some embodiments, the mode of administration is byinjection into the intraocular space or into the vitreous of an eye(e.g., to target ocular tumors or tumors that have metastasized to theeye).

Examples of reagents that may be used to deliver virus-like particles ofthe present disclosure include, without limitation, saline, MgCl₂,trehalose, sodium hyaluronate, polysorbate 20, polysorbate 80 or anycombination of two or more of the foregoing reagents.

Photosensitive molecules of the disclosure can be activated at asuitable wavelength. In some embodiments, activation of thephotosensitive molecules renders them cytotoxic or able to produce acytotoxic molecule. Suitable wavelengths include, without limitation,ultraviolet wavelengths, visible wavelengths, infrared wavelengths andnear infrared wavelengths. In some embodiments, the photosensitivemolecules are activated and become cytotoxic at a wavelength of 600 nmto 800 nm, or 660 nm to 740 nm. In some embodiments, the photosensitivemolecules are activated and become cytotoxic at a wavelength of about600 nm, 610 nm, 620 nm, 630 nm, 640 nm, 650 nm, 660 nm, 670 nm, 680 nm,690 nm, 700 nm, 710 nm, 720 nm, 730 nm, 740 nm, 750 nm, 760 nm, 770 nm,780 nm, 790 nm or 800 nm. In some embodiments, the photosensitivemolecules are activated at a wavelength of less than 600 nm or more than800 nm. Suitable wavelengths for photosensitive molecule activation willdepend on the particular molecule used.

The photosensitive molecules of the disclosure, depending on the type ofmolecule, can be activated by infrared, near-infrared or ultravioletlight. For example, an infrared, near-infrared or ultraviolet laser maybe used, in some embodiments, to activate the photosensitive moleculesof VLP conjugates. The energy delivered by the laser may range fromabout 5 J to about 100 J, about 5 Joules (J) to about 50 J, or about 8 Jto about 36 J. In some embodiments, the energy delivered by the laser is8 J, 9 J, 10 J, 11 J, 12 J, 13 J, 14 J, 15 J, 16 J, 17 J, 18 J, 19 J, 20J, 21 J, 22 J, 23 J, 24 J, 25 J, 26 J, 27 J, 28 J, 29 J, 30 J, 31 J, 32J, 33 J, 34 J, 35 J, 36 J, 37 J, 38 J, 39 J, 40 J, 41 J, 42 J, 43 J, 44J, 45 J, 46 J, 47 J, 48 J, 49 J, 50 J, 51 J, 52 J, 53 J, 54 J, 55 J, 56J, 57 J, 58 J, 59 J, 60 J, 61 J, 62 J, 63 J, 64 J, 65 J, 66 J, 67 J, 68J, 69 J, 70 J, 71 J, 72 J, 73 J, 74 J or 75 J. In some embodiments, theenergy delivered by the laser is 10 J, 20 J, 30 J, 40 J, 50 J, 60 J, 70J, 80 J, 90 J or 100 J.

A light or laser may be applied to the photosensitive molecules (orphotosensitive VLPs) from about 5 seconds to about 5 minutes. Forexample, in some embodiments, the light or laser is applied to thephotosensitive molecules for 5, 10, 15, 20, 25, 30, 35, 40, 45 50 or 55seconds to activate the molecules. In some embodiments, the laser isapplied to the photosensitive molecules for 1, 1.5, 2, 2.5, 3, 3.5, 4,4.5 or 5 minutes, or more. It should be understood that the length oftime a light or laser is applied to a photosensitive molecule can varydepending, for example, on the energy (e.g., wattage) of the later. Forexample, lasers with a lower wattage may be applied to a photosensitivemolecule for a longer period of time in order to activate the molecule.

A light or laser may be applied to the photosensitive molecules (or VLPconjugates) about 30 minutes to about 48 hours after administering theVLP conjugates. For example, in some embodiments, the light or laser isapplied to the photosensitive molecules 30, 35, 40, 45, 50 or 55 minutesafter administering the VLP conjugates. In some embodiments, the lightor laser is applied to the photosensitive molecules 1, 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 or 24 hoursafter administering the VLP conjugates. In some embodiments, the lightor laser is applied to the photosensitive molecules 36 or 48 hours afteradministering the VLP conjugates.

The light or laser may be applied directly to the site of the tumor. Forexample, VLP conjugates targeting ocular tumors may be activated byilluminating the eye.

Any type of tumor can be targeting in accordance with the presentdisclosure. Examples of tumors include, without limitation, thoselocated in the eye, lung, pleura, liver, pancreas, stomach, esophagus,colon, breast, ovary, prostate, brain, meninges, testis, kidneys,bladder, head, neck, cervix, larynx and/or skin.

In some embodiments, the tumor is an ocular tumor. The ocular tumor maybe located in the vitreous, choroidal space, iris, ciliary body, sclera,fovea, retina, optic disk or optic nerve.

The tumor, in some embodiments, is cancerous or malignant. In someembodiments, the tumor is metastatic. Other tumors may also be targeted.For example, the present application provides methods and compositionsfor targeting cervical cancer cells, ovarian cancer cells, melanomacancer cells, lung cancer cells, head and/or neck cancer cells, andbladder cancer cells.

Compositions

The virus-like particles (viral-like nanoparticles) of the presentdisclosure are, in some embodiments, photosensitive molecule-conjugatedviral-like nanoparticles. The viral-like nanoparticles contain one ortwo types of capsid proteins from papilloma virus. In some embodiments,the capsid proteins are modified. Capsid proteins typicallyself-assemble into “empty” proto-capsids approximately 55 nm in diameter(e.g., spherical-like particles containing a hollow core). Aftermaturation of the proto-capsids to form viral-like nanoparticles(virus-like particles), viral-like nanoparticles are then chemicallyconjugated with a photosensitive molecule (e.g., IR700 dye such asIRDye® 700DX, an infrared dye manufactured by LI-COR®).

In some embodiments, the photosensitive viral-like nanoparticles areprovided in a sterile, solution (e.g., 1 or 2 ml) in single use vials(e.g., borosilicate glass vials). In some embodiments, thephotosensitive viral-like nanoparticles are provided in a sterilesolution of water that optionally includes NaCl, KCl, Na₂HPO₄.2H₂O,KH₂PO₄, or any combination of two or more of the foregoing. In someembodiments, NaCl may be present in the solution at a concentration of400 to 600 mMol (e.g., 500 mMol). In some embodiments, KCl may bepresent in the solution at a concentration of 2 to 6 mMol (e.g., 2.7mMol). In some embodiments, Na₂HPO₄.2H₂O may be present in the solutionat a concentration of 5 to 15 mMol (e.g., 10 mMol). In some embodiments,KH₂PO₄ may be present in the solution at a concentration of 1 to 3 mMol(e.g., 2 mMol).

It some embodiments, photosensitive viral-like nanoparticles are dilutedand administered intra-ocularly using a sterile syringe and needlecommonly used in ophthalmic procedures. The present disclosure alsoprovides other routes of administration and administration to othertumors and/or metastases, as described elsewhere herein.

In some embodiments, each viral-like nanoparticle comprises 12-72capsomers with each capsomere containing 5 molecules of L1 capsidprotein (e.g., 55-56 kD each) and 1 molecule of L2 capsid protein (e.g.,52 kD each). In some embodiments, each viral-like nanoparticle comprises12-72 capsomers with each capsomere containing only L1 capsid proteins(e.g., 5 molecules of L1 protein per capsomere).

In some embodiments, each viral-like nanoparticle is chemicallyconjugated (e.g., via an amide bond) with 10 to 1000 molecules (e.g.,500 molecules) of photosensitive molecule (IR700 dye such as IRDye®700DX) to at least one amino acid (e.g., lysine amino acid) of theprotein.

Methods of Producing Virus-Like Particles

To produce photosensitive viral-like nanoparticles of the presentdisclosure, mammalian cells, such as 293T cells (e.g., HEK293F cells)may be grown (e.g., in suspension culture) and transiently transfectedwith a nucleic acid (e.g., bi-cistronic plasmid DNA) encoding BPV or HPVL1 (or L1 and L2) capsid proteins. This induces the formation ofproto-capsids (e.g., as described in Buck et. al. Current Protocols inCell Biology 26.1.1-26.1.19, December 2007). Following cell massrecovery and disruption, the proto-capsids may be subjected to host DNAclearance with benzonase treatment and a subsequent maturation processin vitro to form stable viral-like nanoparticles. Followingpurification, the viral-like nanoparticles may be chemically conjugatedwith photosensitive molecules (e.g., IR700 NHS ester) to produce thephotosensitive viral-like nanoparticles. FIG. 23 shows a schematicrepresentation of an example of a production process provided herein.

Thus, in some aspects, provided herein are methods of producingphotosensitive molecules, comprising (a) transiently transfecting cellswith a nucleic acid that encodes one or more capsid proteins, therebyforming proto-capsids, (b) collecting the proto-capsids and subjectingthe proto-capsids to a maturation process in vitro, thereby formingstable viral-like nanoparticles, and (c) chemically conjugating theviral-like nanoparticles to 50 to 1000 photosensitive molecules. In someembodiments, the viral-like nanoparticles are conjugated to 500photosensitive molecules. In some embodiments, the viral-likenanoparticles are conjugated to photosensitive molecules through anamide bond (e.g., by reacting an ester group of a photosensitivemolecule with an amine group of an amino acid the capsid protein of aviral-like nanoparticle).

EXAMPLES Example 1—Conjugation of IRDye® 700DX

The procedure of chemical conjugation of VLPs (e.g., viral-likenanoparticles containing a combination of variant HPV16/31 L1 proteinsand HPV L2 proteins) to a photosensitive molecule (e.g., IRDye® 700DX)is as follows. Typically, solutions of VLPs were maintained at aconcentration of 1 mg/ml in PBS, pH=7.2 and 0.3 to 0.5 M NaCl. The IR700(e.g., IRDye® 700DX) molecules were supplied from the manufacturer asdry NHS (N-Hydroxysuccinimide) esters (NHS-esters react with aminegroups on proteins to form covalent amide bonds) (FIG. 10A). Availableamine groups on proteins are the amino terminus of the protein or theε-amino group on the amino acid (e.g., lysine). The dry solid IR700-NHSester was dissolved in DMSO at a concentration of 5 mg/ml and storedfrozen. Typically, different ratios of VLP:dye were achieved by mixingdifferent amounts of IR700-NHS to a fixed amount of VLP, usually 1 ml of1 mg/ml solution in PBS. The typical ratios and the amounts of IR700-NHSare listed in the following table:

TABLE 1 Volume of Ratio of Mass of IR700-NHS IR700-NHS solutionIR700-NHS:VLP for 1 mg of VLP for 1 mg of VLP  200:1 16 μg 3.2 μl  500:140 μg   8 μl 1000:1 80 μg  16 μl

To make a 200:1 ratio, 1 ml of VLP at 1 mg/ml in PBS was mixed with 3.2μl of the IR700-NHS ester solution. These reactions were run for 2-4hours at room temperature. Following the completion of the reaction, theVLPs were purified by heparin affinity column chromatography to separatethe unbound IR700-NHS from the newly formed VLP-IR700 conjugate (alsoreferred to as a photosensitive VLP).

Example 2—Conjugation of Visudyne®

The conjugation of Visudyne® to the VLPs followed a slightly differentprotocol relative to the IR700-NHS. Visudyne® molecules requiredfunctionalization to NHS, prior to conjugation to VLPs. Thisfunctionalization was achieved through the use of EDC(1-ethyl-3-(-3-dimethylaminopropyl) carbodiimide hydrochloride). EDC wasused to functionalize molecules that have a free carboxylic acidmolecule, such as Visudyne® (see FIG. 10B; circled), and in the presenceof sulfo-NHS effectively transfer the NHS moiety to this agent. Thisreaction scheme is outlined in FIG. 11. Briefly, approximately 2 mM ofEDC and a 2× molar excess of sulfo-NHS was reacted with varying amountsof Visudyne®. After 15 minutes at room temperature, the reactions werestopped with the addition of 2-mercaptoethanol to a final concentrationof 20 mM. This reaction mixture was added to 1 mg of VLPs at aconcentration of 1 mg/ml in PBS, pH=7.2+0.3-0.5 M NaCl and incubated for2-4 hours at room temperature. Finally, the unreacted components wereseparated from VLP-conjugates by heparin affinity column chromatography.

Example 3—VLP Binding Specificity is Mediated by HSPG and Inhibited byHeparin

SK-OV-3 cells in suspension were treated under the following conditions:no VLP (e.g., viral-like nanoparticles containing a combination ofvariant HPV16/31 L1 proteins and HPV L2 proteins), VLP conjugated toeither Alexa Fluor® 488 (FIG. 3, “AF488*PsV”) or IR700 (FIG. 3,“IR700*PsV”), or the same VLP conjugates incubated in the presence ofHSPG. Following incubation, these cultures were subjected to 4 joules of690 mm near infrared light. A parallel set of non-light irradiated cellsacted as a control. Following irradiation, the cultures were assessedfor the extent of cell death. FIG. 3 shows that the only condition underwhich there was substantial cell killing was cell exposure to IR700*PsVand 4 joules of light. Similar cell death was not observed with exposureto AF488*PsV, revealing that cell death is specific to the IR700 dyeconjugates. Moreover, cell death is almost completely abrogated in thepresence of HSPG, revealing that VLP binding to the cell is critical toIR700-mediated cell death.

Example 4—Cell Death Depends on Infrared Radiation and the Amount of VLPand IR700

SK-OV-3 cells in suspension were treated with differing concentrationsof VLP (e.g., viral-like nanoparticles containing a combination ofvariant HPV16/31 L1 proteins and HPV L2 proteins) had been conjugatedwith differing amounts of IR700 dye (e.g., IRDye® 700DX). VLP withoutconjugation to IR700 dye was used as a control. Following incubation,these cultures were subjected to 0 or 16 joules of 690 nm near-infraredlight. Following the light treatment, the extent of cell death wasassessed. FIG. 4 shows that cell death is dependent on both the presenceof IR700 dye and light treatment. This is supported by the observationthat cell death depends on both VLP concentration and the IR700 dyeconjugation ratio.

Example 5—In Vitro Cell Death of SKOV-3 Cells Upon Irradiation FollowingTreatment with VLPs Conjugated to IR700

SKOV-3 ovarian cancer cells were plated on a 24-well plate and treatedwith two different concentrations of photosensitive VLP particles (e.g.,viral-like nanoparticles containing a combination of variant HPV16/31 L1proteins and HPV L2 proteins conjugated to IR700 dye), 2.5 μg (red) and0.25 μg (blue), for 1 h at 37° C. Upon binding, the cells were washed,followed by treatment with 4 J of light. Cell death was determined uponenzymatic estimation of LDH release (determined by measuring absorbanceat 490 nm). Three different molar ratios of VLP:IR700 conjugation weretested: 1:500, 1:1000 and 1:2000, respectively. FIG. 5 shows thatmaximum efficacy of cell death was observed at a 1:1000 ratio ofVLP:IR700 (“PsV:IR700”) for both concentrations tested.Detergent-mediated cell lysis was used as a positive control.

Example 6—Structural Evaluation of IR700-PsV Complexes

FIG. 6 shows an ESI-TOF analysis of control VLPs (PsV) (A) andIR700-conjugated VLPs (PsVs) (B). In FIG. 6B, the reaction was set up toachieve conjugation of each VLP (PsV) molecule with 1000 molecules ofIR700. The signal spikes in the ESI-TOF scans correspond to the VLP L1protein. A shift of 5517 amu was observed in conjugated samples relativeto control samples, which conjugated samples correspond to an average of3 conjugated IR700 molecules (1840 amu) per L1 protein or about 1000molecules of IR700 per VLP (typically, there are 360 L1 per VLP).

Example 7—Agent Binding Determines Extent of Cell Death in an OcularMelanoma Cell Line

Ocular melanoma cell line (92.1; HER2⁻) in suspension were exposed tovarying dilutions of either Herceptin® antibody conjugated to IR700 dye(e.g., IRDye® 700DX) or VLPs (e.g., viral-like nanoparticles containinga combination of variant HPV16/31 L1 proteins and HPV L2 proteins)conjugated to IR700 dye. Parallel cultures were then assessed for agentbinding (FIG. 7C) or cell death in the absence (FIG. 7B) or presence(FIG. 7A) of 16 joules of 690 nm near-infrared light. FIG. 7C showsconcentration-dependent VLP binding to the 92.1 ocular melanoma cells,while Herceptin® antibody binding is essentially absent. FIG. 7 B showsthat, in the absence of light, there is no cell death. FIG. 7A showsconcentration-dependent cell death only in the photosensitiveVLP-treated cells.

Example 8—Agent Binding Determines Extent of Cell Death in an OvarianCancer Cell Line

SK-OV-3 cells (HER2⁻) in suspension were exposed to varying dilutions ofeither Herceptin® antibodies or VLPs particles (e.g., viral-likenanoparticles containing a combination of variant HPV16/31 L1 proteinsand HPV L2 proteins) conjugated to IR700 dye (e.g., IRDye® 700DX).Parallel cultures were then assessed for Herceptin® or VLP binding (FIG.8C) or cell death in the absence (FIG. 8B) or presence (FIG. 8A) of 16joules of 690 nm near-infrared light. FIG. 8C shows that VLP binding issaturated in SK-OV-3 cells. Herceptin® binding is also concentrationdependent, but to a lesser degree relative to the VLPs. FIG. 8B showsthat, in the absence of light, there is no cell death. FIG. 8A showsconcentration dependent cell death under both conditions, but similar tobinding, the response is saturated with VLPs while there appears to be aconcentration-dependent increase in cell death with Herceptin®. Thesedata imply that the VLPs conjugated to IR700 (PsV-IR700) are more potentthat the Herceptin® conjugated to IR700 (Herceptin-IR700).

Example 9—Vaccine Induced Anti-HPV16 Neutralizing Antibodies do notBlock Binding of BPV*IR700 VLPs to the Ocular Melanoma Cell Line, 92.1

Samples of serum containing different antibodies were tested for theability to inhibit photosensitive VLP particles (e.g., HPV16 VLPs or BPVVLPs) binding to the 92.1 ocular melanoma cell line. FIG. 9 shows that“no serum” or “naïve serum” conditions contain no activity thatneutralizes VLP binding. Moreover, the blocking activity that wasobserved was specific for virus serotype. That is, only human papillomavirus-like particles conjugated to IR700 dye (HPV16-IR700) wereneutralized with serum containing HPV16 antibodies. Bovine papillomavirus-like particles conjugated to IR700 (BPV-IR700) were notneutralized by serum containing HPV16-specific antibodies.

Example 10—Immunogenicity Evaluation

In a study similar to that described in Example 9, neutralizing titerswere determined by serial dilution of sera containing antibodies againsteither HPV16 or BPV. The results described in Table 2 show thatantibodies against HPV16 neutralize only HPV16. Moreover, antibodiesagainst BPV neutralize only BPV. Thus, there is neither cross-reactivityof HVP16 antibodies against BPV nor BPV antibodies against HPV16.

TABLE 2 HPV 16 BPV Human anti-HPV16 1:57,759 1:24 Rabbit anti-HPV161:2,876,000 1:21 Rabbit anti-BPV 1:83 1:14,332

Example 11—Binding Study

The goal of this Example was to assess the binding of viral-likenanoparticles containing human papilloma virus 16 (HPV16) capsidproteins, variant HPV16/31 L1 capsid proteins, and bovine papillomavirus (BPV) capsid proteins to various types of cancer cells. Inaddition, viral-like nanoparticles containing L1 and L2 capsid proteins,or only L1 capsid proteins, were tested to determine if there was adependence on L2 for viral-like nanoparticle binding to cancer cells.Results of this study show that binding of BPV viral-like nanoparticlesand HPV viral-like nanoparticles are comparable.

A large panel of cell lines was screened, which included: miscellaneouscell lines (e.g., 293TT, HaCaT, PAM-212 and TC-1), cervical cell lines(e.g., HeLa, SiHa, CaSki and C-33A), ovarian cell lines (e.g., MOSEC,SHIN-3, SK-OV-3, WF-3, ES-2, A2780, OVCAR-3 and OVCAR-4), melanoma celllines (e.g., B 16F10, SKMEL-2, SKMEL-5, SKMEL-28 and UACC), ocularmelanoma cell lines (e.g., 92.1, MKT-BR, OCM-1 and UW-1), lung celllines (e.g., NCI-H23, NCI-H322M, NCI-H460 and NCI-H522), head and neckcell lines (e.g., CAL-33 (HPV−), FaDu (HPV−), HSC-3 (HPV−), SNU-1076(HPV−), UM-SCC-47 (HPV+), UPCI-SSC-90 (HPV+) and UPCI-SCC-154 (HPV+),and bladder cell lines (e.g., 5637, J82, RT112, SCaBER, SVHUC, T24,UMUC-3, UMUC-5).

Prior to the experiment, viral-like nanoparticles were conjugated toAlexaFluor488 to allow for easy and direct analysis of viral-likenanoparticle binding to the cell surface. AlexaFluor488 was attached tothe viral-like nanoparticle using N-Hydroxysuccinimide (NHS)-esterchemistry, which does not interfere with binding.

Each of the viral-like nanoparticles was tested at a concentration of 10μg/ml, 1 μg/ml and 0.1 μ/ml.

Cells were trypsinized to remove them from the plastic surface of tissueculture plates, washed and allowed to recover for 4 hours at 37° C. ingrowth media on a rocking platform. The cells were then washed, countedand placed into a 96-well round bottom plate at 1×10⁵ cells/well inphosphate buffered saline (PBS)/2% fetal bovine serum (FBS). Theviral-like nanoparticles were added to the cells in a final volume of100 μl PBS/2% FBS. Viral-like nanoparticles pre-incubated with heparin(1 mg/ml, 1 hour, 4° C.) were also added to wells as controls. The cellsand viral-like nanoparticles were then incubated for 1 hour at 4° C. (inthe dark), washed twice with PBS/2% FBS and fixed with 4%paraformaldehyde for 15 minutes at room temperature. Cells were finallywashed again and resuspended in 200 μl PBS/2% FBS and analyzed on a BDFACS CANTO™ II (BD Biosciences, San Jose, Calif.) using BD FACSDIVA™ (BDBiosciences, San Jose, Calif.) and FlowJo software.

Results using the TC-1, HeLa, SK-OV-3, SKMEL-28, 92.1, NCI-H322M, HSC-3,UPCI-SCC-154 and T24 cell lines are presented as histograms in FIG. 12.As evident from FIG. 12, all viral-like nanoparticles, regardless oftheir serotype or makeup (L1 versus L1/L2) bind to cancer cells in thebinding assay. Moreover, heparin competes for binding, demonstratingthat viral-like nanoparticle binding is specific and HSPG dependent.

Example 12—Biodistribution Time Course

The goal of this Example was to assess tumor localization and timecourse of clearance of viral-like nanoparticles following intravenousinjection into tumor-bearing animals.

Purified viral-like nanoparticles were prepared by labeling viral-likenanoparticles (e.g., viral-like nanoparticles containing a combinationof variant HPV16/31 L1 proteins and HPV L2) with IR700 dye (e.g., IRDye®700DX) at a viral-like nanoparticle:dye ratio of 1:500. Thephotosensitive viral-like nanoparticles were purified by densitygradient ultracentrifugation using OPTIPREP™ Density Gradient Medium.

Tumors were generated in albino C57Bl/6 mice by subcutaneous injectionof 2×10⁵ TC-1 cancer cells in 100 μl of PBS. After about two weeks,animals were randomized into treatment groups. Tumor-bearing animalsreceived by intravenous injection either PBS or 200 μg of thephotosensitive viral-like nanoparticles in a volume of 100 μl. Twelve ortwenty-four hours following injection, the animals were euthanized.Following euthanasia, tumor tissue was harvested and imaged forfluorescence of the IR700 dye (e.g., IRDye® 700DX), indicating presenceof the photosensitive viral-like nanoparticles.

FIG. 13B shows detectable IR700 dye (e.g., IRDye® 700DX) fluorescence inthe tumor tissue obtained from both of the 12- and 24-hour time points,while fluorescence in the PBS control (12-hour time point) was notdetected. The quantitative total fluorescence in the tumor tissue isplotted in the graph depicted in FIG. 14.

Example 13—Biodistribution Time Course

The goal of this Example was to assess tumor localization and timecourse of clearance of viral-like nanoparticles following intravenousinjection into tumor-bearing animals.

Purified viral-like nanoparticles were labeled with AlexaFluor488 inlysate and purified by density gradient ultracentrifugation usingOPTIPREP™ Density Gradient Medium.

Tumors were generated in albino C57Bl/6 mice by subcutaneous injectionof 2×10⁵ TC-1 cancer cells in 100 μl of PBS. After about 2 weeks, 200 μgof the photosensitive viral-like nanoparticles were delivered byintravenous injection in a volume of 100 μl. Tumors were harvested atthe following time points following photosensitive viral-likenanoparticle injection: T=1, 2, 4, 8, 12, 24, 48 and 72 hours. Uponharvest, fragments of tumors were frozen for microscopic assessment. Forthis microscopic assessment, tissue sections were further stained.Rabbit polyclonal sera against HPV16 was used in conjunction with anAlexaFluor-488 secondary antibodies. Blood vessels were co-stained witha rat anti-CD31 antibody and an anti-rat AlexaFluor-594 secondaryantibody. Nuclei where highlighted with DAPI.

Data (in situ images not shown) demonstrate the presence of thephotosensitive viral-like nanoparticles at the 1-hour time point. Thelocalization of the signal appeared to be associated within the bloodvessels. The maximum level of staining appeared to occur at the 8-hourtime point, and at the 8-hour time point, the photosensitive viral-likenanoparticles appeared to be diffusing from within the blood vessels tothe tumor cells. Finally, at the 24-hour and 48-hour time points, thereappeared to be little viral-like nanoparticle signal in the tumor.

Example 14—In Vivo Efficacy after Systemic Administration

The study presented in this Example was designed to measure tumorviability 24 hours after a single treatment. The study establishesguidelines for long-term in vivo studies.

Full study design is illustrated in FIG. 15. Due to the range in tumorsizes, animals were randomized such that an even distribution of largeand small tumors were within each group of n=3 in the saline-treatedgroups and n=5 in the IR700 (e.g., IRDye® 700DX)-photosensitiveviral-like nanoparticle-treated groups. Viral-like nanoparticles wereadministered intravenously 12 hours prior to light treatment. Onehundred microgram (100 μg) and 200 μg doses were tested. Light treatmentincluded of 25 J (62.3 s at 400 mW) or 50 J (125 s at 400 mW). After 24hours, tumors were harvested and processed using collagenase and DNaseto generate a single cell suspension. BD LIVE/DEAD® yellow stain wasthen applied, and cells were placed through a FACS CANTO™ II. Data arereported as percentage of dead cells as indicated by a shift influorescence in the Pacific orange channel (FIG. 16A).

A single dose of 200 μg of IR700 (e.g., IRDye® 700DX) photosensitiveviral-like nanoparticles (NPs) was capable of killing the majority ofthe tumor cells after treatment with 50 J of light (FIG. 16B and FIG.17C). The level of killing with 200 μg of NPs was reduced by nearly halfwhen the tumors were treated with 25 J of light (FIG. 16B and FIG. 17B).100 μg of NPs was not enough to induce killing with 25 J of light (FIG.16B and FIG. 17B); however, some tumor death was observed at 50 J dose(FIG. 16B and FIG. 17C). This study provided the necessary IR700 (e.g.,IRDye® 700DX) photosensitive viral-like nanoparticles and light dosageinformation for in vivo studies.

Example 15—Immune System Activation Study

The TC-1 tumor model offers the ability to examine anti-tumor immuneinduction upon treatment with viral-like nanoparticles in immunecompetent animals. The TC-1 tumor line was developed from C57Bl/6 lungepithelial cells immortalized with HPV16 oncogenes E6 and E7 as well asa mutated gene expressing c-Ha-Ras (Lin K Y, et al., Cancer Research.56(1):21-6, 1996). These cells can be implanted subcutaneously or, forstudying metastatic models, they can be injected intravenously to seedcells in the lungs. For nearly twenty years these cells have been usedto test E6 and E7 therapeutic vaccine efficacy. E7 has a distinctive MHCclass I epitope on the C57Bl/6 background that has been shown to beprotective if a CD8 T-cell response can be elicited against it(H-2D^(b), aa 49-57 RAHYNIVTF (SEQ ID NO: 3)) (Feltkamp M C, et al.European Journal of Immunology. 23(9):2242-9, 1993). These responses aredetected by both tetramer staining and re-stimulation of cells with thepeptide followed by intracellular cytokine staining.

Does Response Study: Animals were inoculated subcutaneously with 2×10⁵TC-1 cells in 100 μl of PBS. Approximately two weeks after inoculating,animals were randomized into six groups: (1) no treatment controls, (2)100 μg viral-like nanoparticles (containing a combination of variantHPV16/31 L1 proteins and HPV L2 proteins labeled with IRDye® 700DX)without light controls, (3) PBS with 50 J/cm² light controls, (4) 200 μgviral-like nanoparticles with 50 J/cm² light, (5) 100 μg n viral-likenanoparticles with 50 J/cm² light and (6) 50 μg viral-like nanoparticleswith 50 J/cm² light. Mice received PBS or viral-like nanoparticles byintravenous injection of a 100 μl volume, and light was applied to thetumor 12 hours later using a 690 nm laser. Tumors were harvested 24hours later, digested to generate a single cell suspension, and stainedwith a viability stain to measure the percentage of dead cells (FIG. 18,top).

Several animals in the high dose group experienced symptoms related totumor lysis syndrome, likely due to the massive and rapid tumor necrosisand release of intracellular components into the animals' system. Whilenone of the “100 μg nanoparticles with 50 J/cm² light” group died, micein the group did display some signs of sickness (FIG. 18, top). The “100μg nanoparticles without light” and the “PBS with 50 J/cm² light” groupsdid not display signs of sickness, indicating that the response observedwas due to the combination of the viral-like nanoparticles and light.Overall, necrosis was apparent in all groups that received theviral-like nanoparticles and light. Maximal killing occurred in allgroups, and no dose response was observed (FIG. 18, bottom).

Survival Study: Animals were inoculated subcutaneously with 2×10⁵ TC-1cells in 100 μl of PBS. Approximately two to three weeks afterinoculating, animals were randomized into the treatment group (25 μgviral-like nanoparticles) and the placebo group (PBS only). Micereceived two rounds of treatment, three days apart. A treatment wasconsidered a single intravenous injection of 100 μl of either 25 μg ofviral-like nanoparticles or sterile PBS, followed 12 hours later bylight treatment at 50 J/cm² using a 690 nm laser. Tumor volumes weremeasured every 3-4 days, and animals were euthanized when their tumorsreached a size >1500 mm³ (FIG. 19A).

Treatment with viral-like nanoparticles was able to delay growth oreradiate tumors in animals with tumors less than 500 mm³ (FIGS. 19B and19C). There was no effect on tumor growth kinetics in the placebo group.The two animals that started with the smallest tumors showed no evidenceof tumors within 7 days of the first treatment, and three of the animalsshowed signs of tumor reduction (FIG. 19C).

Immunology Study: For the immunological readout, blood was collected onday 0 (prior to first treatment), day 10 and day 17. Red blood cellswere lysed and the remaining cells were split into two, one half stainedfor cell surface markers (CD62L, CD127, CD103, CD69, CD4, CD8, CD3,H2-D^(b)E7(49-57) tetramer). The other half was re-stimulated for 4.5hours with HPV16 E7 peptide 49-57 followed by staining with antibodiesagainst CD4, CD8 and IFN-gamma as well as a viability dye todiscriminate live cells.

In the blood of the two animals with controlled tumor growth, both “E7tetramer⁺ CD8⁺ T-cells” and “INF-gamma secreting CD8+ cells” (afterre-stimulation with E7 peptide) could be detected, indicating that apotential anti-tumor response had been elicited (FIG. 19C).

Example 16—Histological Analysis

The effects of photosensitive viral-like nanoparticles (e.g., viral-likenanoparticles containing a combination of variant HPV16/31 L1 proteinsand HPV L2 proteins conjugated to IR700 dye) at the histological levelwere assessed using a murine xenograft model. Briefly, 1.5×10⁶ 92.1uveal melanoma cells were implanted into the subcutaneous space of thehind flank of nu/nu mice. The tumors were allowed to reach approximately200 mm³, at which time the animals were treated with an intravenousinjection of 200 μg of photosensitive viral-like nanoparticles. Twelvehours following the injection of photosensitive viral-likenanoparticles, the tumor site was irradiated with 50 J/cm² of 690 nmnear-infrared light. After an additional 24 hours, the animals wereeuthanized, the tumor tissue excised, fixed in formalin, paraffinembedded and processed for standard histological examination.

The hematoxylin and eosin (H&E) images revealed a large degree ofnecrosis, when compared to untreated controls (images not shown). Thetumor treated with photosensitive viral-like nanoparticles and laser hada pale appearance when compared to the control tumor. Upon examinationat higher magnification, the cells of the photosensitive viral-likenanoparticle-treated tumors showed a dramatic loss of cytoplasm comparedto the control treated tumor. Moreover, the extent of necrosis coveredthe entire tumor leading to the conclusion that the NIR light penetratesthrough the entire depth of tumor tissue.

Example 17—Viral-Like Nanoparticle Activity in an Orthotopic XenograftModel of Uveal Melanoma

The most common primary malignancy of the eye is uveal melanoma (UM).Approximately 2,000 patients present annually in the US, with morefrequent occurrence in Europe. Though several treatment options existfor UM, no treatment reliably controls tumor growth, preserves vision,and minimizes the occurrence of radiation-related side effects.

Viral-like nanoparticle phototherapy (PT) is a novel molecular-targetedcancer therapy that involves a two-stage process requiringadministration of both drug and light activation. The drug portion ofviral-like nanoparticle PT is a photosensitive viral-like nanoparticle(NP) conjugated to IRDye®700 DX, a near-infrared (NIR) phthalocyaninedye that acts as a light sensitizer, followed by the application ofnon-thermal NIR light designed to treat adults with primary uvealmelanoma.

In the current study the anti-cancer activity of the photosensitiveviral-like nanoparticle was evaluated in an orthotopic xenograft modelof uveal melanoma. In this model, human uveal melanoma cells wereimplanted into the choroidal space of immunosuppressed rabbits andallowed to grow. When tumors were observable by fundoscopy, the animalswere assigned to treatment or control groups. In both cases, the animalswere followed by fundoscopy and ultrasound for progressive tumor growthor response to treatment. Following termination of the study, thetumor-bearing eyes were also examined by gross and histopathology.

This study was carried out in using 20 total rabbits implanted with the92.1 uveal melanoma cell line. In total, 11 of twenty animals developedtumors. Two animals died unexpectedly during the follow-up period priorto treatment; these animals were used as untreated controls. Severalanimals had extra-ocular tumors that were not lasered; these were usedas internal controls. Animals with tumors in the anterior chamber wereexcluded from the study.

All treated tumors showed a major tumor response compared to controlanimals, demonstrated by fundoscopy, gross pathology and histopathologyevaluation. Retinal tissues adjacent to tumors were not affected by thetreatment.

In conclusion, based on the extent of the tumor response and necrosisseen following photosensitive viral-like nanoparticle administration andlaser administration, the treatment methodology provided herein may beused for the treatment uveal melanoma tumors.

Study Schedule: Two Treatment Groups: 1) Full Tumor Treatment; 2) NoTreatment.

TABLE 3 Animals arrive Apr. 22/23^(rd), 2014 Tumor cell implantationApr. 29/30^(th), 2014 Treatment start May 20^(th), May 27^(th), Jun.3^(rd), 2014 Study termination Jun. 24^(th), 2014 Draft report Jul.25^(th), 2014

Methods and Experimental Design: Test System

TABLE 4 Species: Rabbit Strain: New Zealand Albino Number and Sex Totalordered: 20 Proposed total in study: 20 Sex: F Age at Receipt: 6 monthsSource: Charles River Identification: RFID and ear tag

Model

Cell Culture

Human uveal melanoma cell line 92.1 (courtesy of Dr. Jerry Y.Niederkorn, University of Texas Southwestern Medical Center, Dallas,Tex.) were cultured at 37° C. in 5% CO₂ in complete culture medium(RPMI-1640 with 10% fetal bovine serum, 100 U/mL penicillin G, 250 ng/mLamphotericin B, and 100 μg/mL streptomycin solution).

Animals and Induction of Immunosuppression

New Zealand albino rabbits with a mean initial weight approximately 3 kgwere used for this study. The rabbits were immunosuppressed with dailysubcutaneous injections of cyclosporin A (CsA; Sandimmune 50 mg/mL;Novartis Pharmaceuticals, Cambridge, Mass., USA). CsA administration wasmaintained throughout the experiment to prevent spontaneous tumorregression. The dosage schedule was 15 mg/kg per day for 3 days beforecell inoculation and for 4 weeks thereafter, followed by 10 mg/kg perday until the end of the experiment. Dosage was further attenuated atthe discretion of the veterinarian. CsA doses were adjusted dailyaccording to each animal's body weight. The body weight was measureddaily, and was posted in the room where the rabbits were housed.

During the follow-up, the animals were monitored daily for signs of CsAtoxicity, such as gingival hypertrophy, drooling, diarrhea, and weightloss. If the animals showed early signs of CsA toxicity (e.g. loss ofappetite), the vet staff was consulted immediately for supportivemanagement, such as appetite stimulant and GI motility enhancer.Adjusting the injection dose was also considered according to the vet'srecommendation.

Cell Implantation

On day 3 after CsA treatment, the animals were anesthetized with anintramuscular injection of ketamine (40 mg/kg) and xylazine (6 mg/kg).After anesthesia, 1-3 drops of 0.5% proparacaine hydrochloride wereapplied to the right eyes and 1.0×10⁶ 92.1 human uveal melanoma cells ina volume of 100 μl suspension was injected into the suprachoroidal spaceof the right eye of the rabbits using a bent cannula. Briefly, a steriledrape was placed over the eye in order to avoid any contamination withhairs or eye lashes, and the conjunctiva was cleaned with 10% betadinesolution. Next, the eye was rotated forward using sutures beneath theocular muscles, and after dissecting the conjunctiva, a sclerotomy wasperformed approximately 10 mm from the limbus. The cannula was theninserted into the slerotomy (⅓-½ of its length) and the cells (100 μLcontaining 1.0×10{circumflex over ( )}6 cells) were injected into thesuprachoroidal space. The needle was slowly retracted, and suturesclosing the sclerotomy were tightened to ensure minimal reflux at theinjection site. A drop of antibiotic ophthalmic solution (erythromycinointment) was applied over the surgical wound to prevent infection.

Housing, Feed, Water and Environmental Conditions, Acclimation

The animals were housed in group housing in groups of 6 and fed foodthat is fresh, palatable, and nutritionally adequate ad libitum. Waterthat is clean, potable, and uncontaminated was provided ad libitum.Environmental controls were set to maintain temperatures 22±4° C. (68±5°F.) with relative humidity of 50%±20%. A 12-hour light/dark cycle wasmaintained. The animals were acclimated for at least 5 days afterarrival at the facility prior to baseline evaluation. Animals wereassigned to test groups after baseline fundoscopic evaluations.

Test and Control Articles

TABLE 5 Vehicle of Test Article Identity: PBS Storage Conditions: 4° C.for up to 3 months protected from light Handling Precautions: StandardPPE

TABLE 6 Test Article Identity: Nanoparticle labeled with IRDye ® 700 DXStorage Conditions: 4° C. for up to 2 months protected from lightHandling Precautions: Standard PPE

TABLE 7 Laser Power Setting:  600 mW Duration:   83 s Fluence:   50J/cm² Spot size:  5.0 mm Wavelength:  690 nm

Preparation of Dose Formulations

The test article was diluted 1:1 in sterile water for injection.

Administration of Test/Control Articles Dosing: Photosensitiveviral-like nanoparticle or saline was administered by intraocularinjection in the vitreous.

Laser administration: Laser treatment was applied using a slit lampsystem with a Coherent Opal Photoactivator® laser delivering 690 nmlight at a power of 600 mW over a duration of 83 seconds for a totalfluence of 50 J/cm². The laser spot size was set to a diameter of 5 mmand as such, tumors that were greater than this size were lasered withoverlapping spots. In cases where a clear distinction of the tumorborder could not be delineated due to ocular complications (e.g.,vitritis, retinal detachment) the entire suspicious area was lasered.

Mortality/Morbidity Checks: All animals remained in good healththroughout the study, aside from the two animals that died due to CsAcomplications (see below).

Clinical Observations: All animals were observed daily by animalfacility personnel; observations were recorded. Most animals experiencedsome degree of weight loss and loss of appetite, which was attributed tothe CsA.

Ophthalmology

Frequency: Ophthalmic examination by fundoscopy and ultrasound wasperformed weekly.

Procedure: The animal was sedated and their right eye was dilated usingocular phenylephrine hydrochloride and tropicamide drops. Next, afundoscopic examination of the eye was performed using an indirectbinocular ophthalmoscope. The ophthalmologist recorded any ocularcomplications. When a tumor was identified, the size was estimated bycomparing it to the optic disc (disc diameter [DD]; 1 DD=approximately1.75 mm). For the ultrasound readings, immediately following fundusexam, an ultrasound probe was applied to the eye to visualize thelocation of the tumor as determined by fundus exam. Ultrasoundmeasurements proved technically difficult, primarily because some of thetumors were located too peripherally to be properly visualized. As aresult, it was not always possible to measure the largest tumordimension; and in most cases only the height in was quantifiable.

Terminal Procedures and Anatomic Pathology

Unscheduled Deaths: Of the 20 animals used in this study, one waseuthanized due to weight loss (>20% of weight upon arrival), as per theprotocol guidelines. One animal died unexpectedly due togastrointestinal stasis caused by CsA toxicity.Scheduled Euthanasia: Upon the termination of the study, animals wereeuthanized in accordance with accepted American Veterinary MedicalAssociation (AVMA) guidelines. The animals were exsanguinated withanesthesia using a combination of ketamine-xylazine-acepromazine (0.75mg/kg, 5 mg/kg, and 20-35 mg/kg, respectively) and buprenorphine (0.2mg/kg).

Results

Overall, 11 animals developed histopathologically evident tumors. Aspreviously mentioned, two animals that died unexpectedly and were usedas the untreated control. 9 animals with different tumor sizes receivedtreatment with photosensitive viral-like nanoparticles. One animal wasnot included in the evaluation due to the extent of tumor in theanterior segment of the equator that could not be lasered.

For animals that had tumors in the back of the eye and received fulltreatment (photosensitive viral-like nanoparticle+laser) a noticeabletumor response was observed, which was characterized by threeelements: 1) induction of extensive tumor necrosis; 2) change in thegrowth pattern, from diffuse to a “sleeve-like pattern”; and 3) sparingof the adjacent retina.

TABLE 8 Fundoscopy Fundoscopy and/or and/or ultrasound: ultrasound:presence tumor Animal number of tumor evaluation & tumor location beforeafter Gross & and size treatment treatment histopathology 3-Largetumor + Tumor Intraocular tumor with posterior to the growth overallnecrotic equator arrest consistency on gross pathology >50% necrosis byhistopathology No damage to adjacent retina 6-Large tumor + TumorIntraocular tumor posterior to the growth disaggregated on equatorarrest gross pathology evaluation >70% necrosis by histopathology Nodamage to adjacent retina 7-Medium size + Complete Non-pigmented tumortumor posterior to response on gross pathology the equator No tumorfound on histopathology- suspicious area ~8.2 mm in length suspected tocorrespond to tumor location 9-Small tumor + Complete No tumor on grossposterior to the response pathology equator Complete response onhistopathology; no damage to adjacent retina 10-Medium sized + TumorNon-pigmented tumor tumor posterior to shrinkage located at the equatorthe equator on gross pathology ~70% necrosis on gross pathology 11-Smalltumor + Complete No tumor on gross or posterior to the responsehistopathology equator 15-Large tumor + Complete Non-pigmented tumorposterior to the response on gross pathology equator of treated locatedat the nodule periphery and Partial additional adjacent necrosis intumor nodule on the peripheral posterior of the eye tumor Histopathologyrevealed no tumor where the treated nodule was present. Other areas thatwere too peripheral were difficult to have full access with the laserand show some extent of necrosis at the apex of the tumor 16-Tumor +Complete Non-pigmented posterior to the response nodule identified onequator gross pathology No tumor on histopathology; scar (fibrosis andinflammation) measuring ~1.5 mm in the area where the tumor is believedto have been located 19-Large tumor + Tumor Large tumor on grossposterior to the growth pathology with equator arrestdisaggregated/necrotic appearance Massive necrosis, sleeve-like pattern

Untreated Controls

Rabbit #14 was euthanized on week 4 due to unacceptable weight loss(>20% of initial body weight). Fundus examination for the presence oftumor was inconclusive due to massive hemorrhage and retinal detachment.This animal received no treatment.

Ultrasound: This rabbit did not undergo an ultrasound examination due tothe timing of death.

Gross/histopathology: On gross pathology, an intraocular tumor measuring3 mm in height (H)×8 mm in the largest tumor dimension (LTD), and onhistopathology an intraocular tumor measuring 2.2 mm H and 9.5 inlargest tumor dimension, was noted. On gross pathology, an extraoculartumor measuring 1.4 mm in height and 9.4 mm in largest tumor dimensionwere noted. Approximately 10% of both the intra- and extraocular tumorswere necrotic. No sleeve pattern was detected.

Full Treatment

Rabbit 9

Rabbit #9 had a clinically detectable tumor on fundus approximately 1 DDin size on week 4, which was treated immediately. The following week,the tumor was estimated as 0.5 DD. On week 6, the tumor was estimated at<0.5 DD and on the final week it was not detected.

Ultrasound: Ultrasound did not discern a tumor on week 3, but on weight4 a mass measuring 1.04 mm in height was identified. On subsequentweeks, the measurements on ultrasound regressed until it was no longervisible by week 6 and thereafter.

Gross/Histopathology: Histopathology revealed no tumor or cells.However, serial sections of the entire eye and immunohistochemicalresults are pending to further confirm this result.

Rabbit 6

There was a clinical suspicion of a tumor on week 4 (elevated massbeneath the retina), but subretinal hemorrhage, fluid, and retinaldetachment precluded clinical size estimation for the duration of theexperiment.

Ultrasound: By ultrasound, on week 3 a large 4.88 mm mass was detected,which grew to 5.29 mm on week 4, at which point we commenced treatment.On week 5, the tumor measured 4.88 mm, while on weeks 6 and 7 the tumormeasured 4.02 and 4.98 mm, respectively.

Gross/histopathology: On gross pathology, two distinct tumors wereidentified: an intraocular tumor and a conjunctival tumor, the lattersuspected to be a result of reflux during cell implantation. Owing tothe location of the conjunctival tumor, it was not treated, and thus weconsider it as an internal control. The intraocular tumor wasdisaggregated and measured 7 mm H×11 mm LTD. An extraocular extensionmeasuring 6 mm H×9 LTD mm was also identified, which had acharacteristic texture. On histopathology, the intraocular tumormeasured 4.9 mm H×8.3 LTD mm and was >70% necrotic, with most of theremaining viable cells forming the aforementioned sleeve-like pattern.The untreated conjunctival tumor exhibited far less necrosis (˜15%) andthe sleeve pattern was not evident.

The goal of this study was to explore the activity of photosensitiveviral-like nanoparticles+NIR light in an orthotopic xenograft model ofuveal melanoma in the rabbit eye. All tumors that receivedphotosensitive viral-like nanoparticles+laser treatment respondedfavorably to the treatment. This is particularly evident for small tomedium tumors that had evident tumor shrinkage as a response oftreatment and complete histopathological responses. In rabbit 9, forexample, that presented with a small tumor at week 4, the tumor wascompletely eradicated by the first two doses of the treatment and was nolonger detectable either clinically or histopathologically two weeksafter the second treatment. In bigger tumors, for example rabbit 4, mostof the tumor was necrotic, this is in stark contrast to the untreatedcontrol (rabbit 14), which only exhibited necrosis in approximately 10%of the tumor volume, a clear indication of the efficacy of thetreatment. Moreover, several animals that had intraocular tumors thatreceived full treatment had extraocular extensions that were notlasered; these fractions showed substantially less necrosis than treatedtumor fractions, which is further evidence supporting the efficacy oflaser activated photosensitive viral-like nanoparticles for thetreatment of uvea melanoma. Retinal areas adjacent to tumors were notaffected by the treatment.

Based on the intraocular tumor response following treatment, especiallycompared to the controls (untreated, extraocular fractions), the dataprovided herein support a selective and potent anti-cancer activity forphotosensitive viral-like nanoparticles, in the presence of a tumor, forthe treatment of ocular melanoma.

Example 18—In Vitro Potency Assay Comparing HPVL1 vs BPV L1

Photosensitive viral-like nanoparticles (e.g., viral-like nanoparticlescontaining a combination of variant HPV16/31 L1 proteins and HPV L2proteins conjugated to IR700 dye) potency was assayed by an in vitrocell killing assay. Uveal melanoma cells (e.g., cell line OCM-1 or 92.1)were harvested by routine methods using a solution of EDTA and trypsin.Once removed from the tissue culture plastic, the cells were suspendedin complete growth media and were allowed to recover for approximately30 minutes at 37° C. During this recovery period, serial dilutions ofthe photosensitive viral-like nanoparticle were made in approximately ½log increments (2000 pM, 600 pM, 200 pM, 60 pM, 20 pM, 6 pM, 2 pM and0.6 pM) in PBS+2% fetal bovine serum. Following the recovery period, thecells were counted, centrifuged and suspended in PBS+2% FBS to a celldensity of 3×10⁶/ml. An equal volume of cell suspension was added to theviral-like nanoparticle dilutions to yield 1.5×10⁶ cells per ml in theappropriate concentration (1000 pM, 300 pM, 100 pM, 30 pM, 10 pM, 3 pM,1 pM and 0.3 pM) of viral-like nanoparticle. These conditions (e.g., 360μl) were incubated on ice for about 1.5 to 2 hours.

Following this incubation, the tubes were centrifuged to collect thecells and the cells were subsequently washed twice with PBS+2% FBS,without the photosensitive viral-like nanoparticles. After the finalcentrifugation, the cells were suspended in 200 μl of PBS+2% FBS. A 100μl of each sample is removed and transferred to the well of a 96-well, ½area plate. Each sample was then irradiated with 25 J/cm2 (600 mW, 43seconds) of near infrared light (689 nm) using a Coherent OpalPhotoactivator ophthalmic laser. Following the irradiation, the sampleof cells was then transferred to a new tube. Both the light irradiatedand non-irradiated samples were placed at 37° C. for an additional 1 to2 hours.

Following this incubation a final 20 μl sample of cells were mixed 1:1with AOPI stain (Acridine Orange and Propidium Iodide) and the viabilityof the cells was evaluated using a Nexcelom Cellometer Auto 2000. FIG.20 shows comparable effects of BPVL1 and HPVL1 on cell viability at halfmaximal effective concentration (EC50) (BPVL1=88 pm; HPVL1=60.5 pm),indicating that the potencies of the photosensitive molecules arecomparable to each other.

FIG. 21 shows a sample of cells from the killing assay described in FIG.20 analyzed for photosensitive viral-like nanoparticle binding. Cellsfrom the killing assay were scanned on an Odyssey Clx gel/plate scanner.The Odyssey Clx is specifically designed for the detection andquantitation of a series of infrared dyes, including, IR700 dye (e.g.,IRDye® 700DX). Thus, in this assay, the cells that were treated withdifferent concentrations of the photosensitive viral-like nanoparticlesshow a concentration dependent amount of fluorescence associated withthe cells, indicating that the cells bound both BPV-L1-IR700 andHPV-L1-IR700.

Example 19—Activity of Photosensitive Viral-Like Nanoparticles in aXenograft Model of Head and Neck Cancer

Head & Neck cancer cells were implanted in the dorsal lateral flank ofnu/nu mice. Tumors were allowed to grow for two weeks. Once the tumorreached an average size of 150 mm³, the animals were randomized into 6study groups (7 animals per group), as follows: Saline; photosensitiveviral-like nanoparticles (HPV16/31 L1/L2; 200 μg dose); Saline+NIR light(50 J/cm2); photosensitive viral-like nanoparticles (200 μg dose)+NIRlight (50 J/cm2); photosensitive viral-like nanoparticles (100 μgdose)+NIR light (50 J/cm2); and photosensitive viral-like nanoparticles(50 μg dose)+NIR light (50 J/cm2). Dosing and NIR light treatment wasperformed every three days. Tumor measurements were recorded every 3-5days.

While all the controls showed no substantial effect for their respectivetreatments, there was a significant tumor growth inhibition observed inall of the dose groups (FIG. 22). The observed tumor growth inhibitionwas dose dependent. There was a response of the tumors in the high dosegroup. Two animals died in the 200 μg treatment group associated withmassive cell death and potentially Tumor Lysis Syndrome relatedtoxicities.

Example 20—Production of Photosensitive Viral-Like Nanoparticles

To produce photosensitive viral-like nanoparticles of the presentdisclosure, HEK293F were grown in suspension culture and weretransiently transfected with a bi-cistronic plasmid DNA encoding L1 (orL1 and L2) capsid proteins. This induces the formation of proto-capsids(as described in Buck et. al. Current Protocols in Cell Biology26.1.1-26.1.19, December 2007). Following cell mass recovery anddisruption, the proto-capsids went through benzonase treatment toeliminate the host DNA contaminants and a subsequent maturation processin vitro to form viral-like nanoparticles stable for conjugation.Following purification, the viral-like nanoparticles were chemicallyconjugated with IR700 NHS ester to produce the photosensitive viral-likenanoparticles. FIG. 23 shows a schematic representation of an aproduction process.

Photosensitive viral-like nanoparticles produced from the processdescribed in this Example have been characterized using SDS-PAGE,SE-HPLC and DLS and show purities of 90-95%. Histones from the HEK293cells are present as part of the viral-like nanoparticle composition andcomprise of 10-15% of the total protein of the viral-like nanoparticle.

Sequences Variant HPV 16/31 L1 protein nucleotide sequence(SEQ ID NO: 1) ATGAGCCTGTGGCTGCCCAGCGAGGCCACCGTGTACCTGCCCCCCGTGCCCGTGAGCAAGGTGGTGAGCA CCGACGAGTACGTGGCCAGGACCAACATCTACTACCACGCCGGCACCAGCAGGCTGCTGGCCGTGGGCCA CCCCTACTTCCCCATCAAGAAGCCCAACAACAACAAGATCCTGGTGCCCAAGGTGAGCGGCCTGCAGTAC AGGGTGTTCAGGATCCACCTGCCCGACCCCAACAAGTTCGGCTTCCCCGACACCAGCTTCTACAACCCCG ACACCCAGAGGCTGGTGTGGGCCTGCGTGGGCGTGGAGGTGGGCAGGGGCCAGCCCCTGGGCGTGGGCAT CAGCGGCCACCCCCTGCTGAACAAGCTGGACGACACCGAGAACGCCAGCGCCTACGCCGCCAACGCCGGC GTGGACAACAGGGAGTGCATCAGCATGGACTACAAGCAGACCCAGCTGTGCCTGATCGGCTGCAAGCCCC CCATCGGCGAGCACTGGGGCAAGGGCAGCCCCTGCACCAACGTGGCCGTGAACCCCGGCGACTGCCCCCC CCTGGAGCTGATCAACACCGTGATCCAGGACGGCGACATGGTGGACACCGGCTTCGGCGCCATGGACTTC ACCACCCTGCAGGCCAACAAGAGCGAGGTGCCCCTGGACATCTGCACCAGCATCTGCAAGTACCCCGACT ACATCAAGATGGTGAGCGAGCCCTACGGCGACAGCCTGTTCTTCTACCTGAGGAGGGAGCAGATGTTCGT GAGGCACCTGTTCAACAGGGCCGGCGCCGTGGGCGAGAACGTGCCCACCGACCTGTACATCAAGGGCAGC GGCAGCACCGCCACCCTGGCCAACAGCAACTACTTCCCCACCCCCAGCGGCAGCATGGTGACCAGCGACG CCCAGATCTTCAACAAGCCCTACTGGCTGCAGAGGGCCCAGGGCCACAACAACGGCATCTGCTGGGGCAA CCAGCTGTTCGTGACCGTGGTGGACACCACCAGGAGCACCAACATGAGCCTGTGCGCCGCCATCAGCACC AGCGAGACCACCTACAAGAACACCAACTTCAAGGAGTACCTGAGGCACGGCGAGGAGTACGACCTGCAGT TCATCTTCCAGCTGTGCAAGATCACCCTGACCGCCGACGTGATGACCTACATCCACAGCATGAACAGCAC CATCCTGGAGGACTGGAACTTCGGCCTGCAGCCCCCCCCCGGCGGCACCCTGGAGGACACCTACAGGTTC GTGACCAGCCAGGCCATCGCCTGCCAGAAGCACACCCCCCCCGCCCCCAAGGAGGACCCCCTGAAGAAGT ACACCTTCTGGGAGGTGAACCTGAAGGAGAAGTTCAGCGCCGACCTGGACCAGTTCCCCCTGGGCAGGAA GTTCCTGCTGCAGGCCGGCCTGAAGGCCAAGCCCAAGTTCACCCTGGGCAAGAGGAAGGCCACCCCCACC ACCAGCAGCACCAGCACCACCGCCAAGAGGAAGAAGAGGAAGCTGTGA BPV1 L1 nucleotide sequence (SEQ ID NO: 2)ATGGCCCTCTGGCAGCAGGGGCAGAAACTCTACCT GCCACCCACACCCGTGTCAAAAGTCCTGTGTTCCGAGACATACGTCCAGCGGAAGTCAATCTTCTACCAC GCCGAGACCGAAAGGCTCCTCACCATCGGCCACCCCTACTACCCCGTCAGCATTGGCGCTAAGACCGTGC CCAAAGTCTCCGCCAACCAATACCGCGTGTTCAAGATCCAGCTGCCCGACCCAAACCAGTTCGCCCTGCC CGATCGCACCGTGCATAACCCCTCCAAGGAAAGACTCGTCTGGGCCGTGATCGGCGTCCAAGTCTCACGG GGCCAACCCCTGGGCGGCACCGTGACCGGCCATCCAACCTTCAACGCCCTCCTGGACGCCGAGAACGTCA ACCGGAAAGTCACAACACAAACCACCGACGATCGCAAGCAGACCGGGCTGGACGCCAAACAGCAGCAAAT CCTCCTCCTGGGGTGCACACCCGCTGAGGGCGAGTACTGGACCACCGCTCGGCCCTGCGTGACCGACAGG CTGGAGAACGGGGCTTGTCCCCCCCTGGAGCTGAAGAATAAGCATATCGAGGACGGCGACATGATGGAGA TCGGCTTCGGCGCCGCTAACTTCAAGGAGATCAACGCCTCCAAGAGCGACCTGCCCCTGGATATCCAGAA CGAAATTTGTCTCTATCCCGATTATCTGAAGATGGCCGAAGATGCCGCCGGCAACTCAATGTTTTTCTTC GCCCGCAAGGAGCAAGTCTACGTGCGGCATATTTGGACACGGGGCGGGAGCGAAAAGGAGGCTCCCACAA CCGACTTCTACCTGAAAAACAACAAGGGCGACGCTACACTGAAGATCCCATCCGTCCACTTCGGCTCCCC ATCCGGGAGCCTCGTCAGCACCGACAACCAGATCTTCAACAGACCATATTGGCTGTTTAGGGCTCAAGGG ATGAATAACGGCATCGCTTGGAACAACCTGCTCTTCCTGACCGTCGGCGATAACACCAGGGGCACCAACC TGACAATCTCCGTGGCTAGCGACGGCACACCCCTGACCGAATACGACTCAAGCAAGTTTAACGTGTATCA CCGGCACATGGAGGAGTACAAACTGGCTTTCATCCTGGAACTGTGTAGCGTCGAGATTACCGCCCAGACC GTCAGCCACCTCCAGGGCCTGATGCCAAGCGTCCTGGAGAACTGGGAGATCGGCGTCCAACCACCAACAA GCAGCATCCTGGAAGATACATACAGATACATCGAAAGCCCCGCCACCAAGTGCGCCTCAAACGTGATCCC CGCCAAGGAGGATCCCTACGCCGGCTTCAAATTCTGGAATATCGACCTGAAGGAGAAACTGAGCCTCGAT CTGGACCAGTTCCCACTCGGCCGGCGGTTCCTGGCCCAACAGGGCGCTGGCTGCAGCACCGTCCGGAAGA GGCGGATCTCACAAAAGACCAGTTCCAAACCCGCCAAGAAGAAGAAGAAGTAG

1.-83. (canceled)
 84. A method comprising: administering to a subject avirus-like particle (VLP) comprising viral capsid proteins and about 50to about 1000 molecules that are activated by infrared, near-infrared,or ultraviolet light, wherein the subject has cancerous cells; andapplying to the cancerous cells a light that activates the molecules.85. The method of claim 84, wherein the molecules comprise dye moleculesthat are activated by infrared, near-infrared, or ultraviolet light. 86.The method of claim 84, wherein the molecules are conjugated to theviral capsid proteins.
 87. The method of claim 86, wherein molecules arecovalently conjugated to lysine residues in the viral capsid proteins.88. The method of claim 87, wherein the molecules are covalentlyconjugated to lysine residues in the viral capsid proteins through anamide bond.
 89. The method of claim 84, wherein the VLP comprises about100 to about 1000 molecules that are activated by infrared,near-infrared, or ultraviolet light.
 90. The method of claim 84, whereinthe VLP comprises about 50 to about 500 molecules that are activated byinfrared, near-infrared, or ultraviolet light.
 91. The method of claim84, wherein the VLP comprises about 100, about 200, or about 300molecules that are activated by infrared, near-infrared, or ultravioletlight.
 92. The method of claim 84, wherein the molecules are activatedby infrared light.
 93. The method of claim 84, wherein the molecules areactivated by near-infrared light.
 94. The method of claim 84, whereinthe molecules are activated by ultraviolet light.
 95. The method ofclaim 84, wherein the VLP has a diameter of 20 to 60 nm.
 96. The methodof claim 84, wherein the administering comprises injecting the VLP intothe subject.
 97. The method of claim 84, wherein the cancerous cellsform a tumor, and the administering comprises injecting the VLP into thetumor.
 98. The method of claim 84, wherein the cancerous cells are in aneye, lung, pleura, liver, pancreas, stomach, esophagus, colon, breast,ovary, prostate, brain, meninges, testis, kidney, bladder, head, neck,cervix, larynx, or skin of the subject.
 99. The method of claim 84,wherein the applying is 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6hours, 7 hours, 8 hours, 9 hours, 10 hours, 11 hours, 12 hours, 13hours, 14 hours, 15 hours, 16 hours, 17 hours, 18 hours, 19 hours, 20hours, 21 hours, 22 hours, 23 hours, 24 hours, 2 days, or 3 days afterthe administering.
 100. The method of claim 84, wherein the applying isfor about 5 seconds to about 5 minutes.
 101. A method comprising:injecting into a tumor of a subject a virus-like particle (VLP)comprising viral capsid proteins and about 100 to about 500 moleculesthat are activated by infrared, near-infrared, or ultraviolet light,wherein the molecules are conjugated to the viral capsid proteins; andapplying to the tumor a light that activates the molecules.
 102. Themethod of claim 101, wherein the light has a wavelength of about 700 nm.103. The method of claim 101, wherein the light has a wavelength ofabout 800 nm.
 104. The method of claim 101, wherein the molecules arecovalently conjugated to lysine residues in the viral capsid proteinsthrough an amide bond.
 105. The method of claim 101, wherein the tumoris in an eye, lung, pleura, liver, pancreas, stomach, esophagus, colon,breast, ovary, prostate, brain, meninges, testis, kidney, bladder, head,neck, cervix, larynx, or skin of the subject.
 106. A method comprising:administering to a subject a virus-like particle (VLP) comprising viralcapsid proteins and about 100 to about 500 molecules that are activatedby infrared or near-infrared light, wherein the molecules are conjugatedto the capsid proteins; and exposing the VLP to an infrared ornear-infrared light.
 107. The method of claim 106, wherein the light hasa wavelength of about 700 nm.
 108. The method of claim 106, wherein thelight has a wavelength of about 800 nm.
 109. The method of claim 106,wherein the molecules are covalently conjugated to lysine residues inthe capsid proteins through an amide bond.
 110. The method of claim 106,wherein the subject has cancerous cells.
 111. The method of claim 106,wherein the subject has a tumor.